Cart protein and DNA encoding therefor

ABSTRACT

Disclosed are DNA sequences encoding to cocaine and amphetamine regulated (CART) proteins, polypeptide products of recombinant expression of these DNA sequences, peptides whose sequences are based upon the amino acid sequences deduced from these DNA sequences, antibodies specific for such proteins and peptides, procedures for the detection and quantitation of such proteins and nucleic acids related thereto, as well as procedures relating to the development of bacteriolytic methods, therapeutic agents, and compositions utilizing CART proteins.

This invention was funded in part by the National Institutes of Healththrough the National Institute on Drug Abuse under Grant No. RO1DA04154. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to Cocaine and AmphetamineRegulated Transcript (CART) proteins and more particularly to mammalianCART proteins, to DNA sequences encoding CART proteins, to thepolypeptide products of recombinant expression of these DNA sequences,to peptides whose sequences are based upon the amino acid sequencesdeduced from these DNA sequences, to antibodies specific for suchproteins and peptides, to procedures for the detection and quantitationof such proteins and nucleic acids related thereto, as well as toprocedures relating to the development of therapeutic agents utilizingCART proteins.

BACKGROUND OF THE INVENTION

Cocaine is a psychomotor stimulant affecting mammalian physiology andbehavior following both acute and chronic patterns of administration.These adaptive changes result in the establishment of physical statesrepresenting tolerance, dependence, sensitization and withdrawal.Pharmacologically, the drug acts by inhibiting the synaptosomal uptakeof catecholamines (including dopamine and norepinephrine), and serotonin(Gawin, "Cocaine Addiction: Psychology and Neurophysiology," Science,251:1580-1586 (1991); Koob, "Drugs of Abuse: Anatomy, Pharmacology andFunction of Reward Pathways," Trends in Neuroscience, 13:177-184(1992)). Modulation of dopaminergic neurotransmission within thestriatum, for example, is believed to underlie the rewarding andreinforcing properties associated with cocaine administration (Kuhar etal, "The Dopamine Hypothesis of the Reinforcing Properties of Cocaine"Trends in Neuroscience, 14:299-302 (1991)).

It is currently hypothesized that cellular plasticity within specificneural circuits underlies the behavioral and physiological alterationsassociated with psychomotor stimulant administration (for review, seeNestler, "Molecular mechanisms of drug addiction," J Neuroscience12:2439-2450 (1992)). One such type of plasticity occurs at the nuclearlevel, and involves the regulated expression of specific sets of genes.For example, cocaine selectively regulates the pattern of expression ofimmediate early genes (IEGs), particularly those belonging to the Fosand Jun (i.e., AP-1) family of transcriptional regulatory factors,within the brain. Furthermore, such transcriptional regulation appearsto be localized to those brain regions regulated by catecholaminergicinput. For example, it is now firmly established that acuteadministration of cocaine induces expression of c-fos and jun B mRNA inthe rat striatum (Graybiel et al., "Amphetamine and Cocaine InduceDrug-Specific Activation of the c-fos Gene in Striosome-MatrixCompartments and Limbic Subdivisions of the Striatum," Proc. Natl. Acad.Sci. USA, 87:6912-6916 (1990); Dragunow et al., "3,4MethyleneDioxyMethamphetamine Induces Fos-like Proteins in Rat BasalGanglia: Reversal With MK-801," Eur. J Pharmacol., 206:255-258 (1991);Hope et al., "Regulation of Immediate Early Gene Expression and AP-1Binding in the Rat Nucleus Accumbens by Chronic Cocaine," Proc. Natl.Acad. Sci. USA, 89:5764-5768 (1992); Nguyen et al., "DifferentialExpression of c-Fos and Zif-268 in Rat Striatum After Haloperidol,Clozapine and Amphetamine," Proc. Natl. Acad. Sci. USA, 89:4270-4274(1992); Young et al., "Cocaine Induces Striatal c-fos-ImmunoreactiveProteins via Dopaminergic D1 Receptors," Proc. Natl. Acad. Sci. USA,88:1291-1295 (1991)), a brain structure regulated by catecholaminergicinput and representing a crucial component of the neuronal circuitryunderlying reward. The cerebellum also represents a brain structure inwhich c-fos mRNA levels are selectively elevated following acute cocaineadministration (Iadarola et al., "Induction and Suppression ofProto-Oncogenes in Rat Striatum After Single and Multiple Treatmentswith Cocaine and GBR-12909," In NIDA research monograph series:Activation of immediate early genes by drugs of abuse (Grzanna et al.,Eds.), 125:181-211 (1993); Clark et al., "Expression of c-fos mRNA inAcute and Kindled Cocaine Seizures in Rats," Brain Res., 582:101-106(1992)). Studies have further shown that cerebellar c-fos mRNA levelsrapidly increase following acute cocaine treatment (Iadarola et al.,supra; Clark et al., supra), with chronic treatment resulting in asensitization of the transcriptional response (Couceyro et al., "CocaineDifferentially Regulates Activator Protein-i 1 mRNA Levels and DNABinding Complexes in the Rat Striatum and Cerebellum," Mol. Pharmacol.,46:667-676 (1994)). Thus, transcriptional plasticity appears torepresent a cellular mechanism through which specific neural networksrespond and adapt to psychomotor stimulant administration. By contrast,the hippocampus represents a transcriptionally quiescent brain structurefollowing acute administration of psychomotor stimulants. Thus, suchbrain region specific transcriptional changes associated withpsychomotor stimulant administration are likely to be related toreinforcement and addiction.

An important area of current research involves identification ofadditional psychomotor stimulant regulated genes. Identification of suchgenes will increase our understanding of the molecular events underlyingboth short- and long-term cellular changes resulting from administrationof psychomotor stimulant drugs, and may potentially lead to thedevelopment of therapeutic agents which mediate effects of psychomotordrugs.

SUMMARY OF THE INVENTION

In accordance with the present invention, differential display PCRtechniques (Liang and Pardee, "Differential Display of EukaryoticMessenger RNA by Means of the Polymerase Chain Reaction," Science,257:967-971 (1992); Liang et al., "Distribution and Cloning ofEukaryotic mRNAs by Means of Differential Display: Refinements andOptimization," Nucleic Acids Res., 21:3269-3275 (1993); Bauer et al.,"Identification of Differentially Expressed mRNA Species by an ImprovedDisplay Technique (DDRT-PCR)," Nucleic Acids Res., 21:4272-4280 (1993))were used to obtain a cDNA representing a rat mRNA whose striatal levelsare increased 4- to 5-fold following acute administration of cocaine oramphetamine. This mRNA (termed CART; Cocaine and Amphetamine RegulatedTranscript) is either 700 or 900 bases in length depending on the siteof poly(A) addition. A presumed alternate splicing event furthergenerates diversity within the rat CART transcripts, and results in thepresence or absence of an in-frame 39 base insert within the putativeprotein coding region. As a result, the predicted translation productsare either 129 or 116 amino acids in length. Northern blot analysis ofvarious rat tissues has determined that CART mRNA is expressedexclusively in neural and endocrine tissues, thus suggesting afunctional role for the predicted protein within the neuroendocrinesystem. Features of the predicted protein include a prototypic signalsequence, suggesting that the protein is targeted for entry into thesecretory pathway and thus serving as a neuroendocrine signalingmolecule.

In addition to the rat CART cDNA and encoded CART protein, correspondinghuman hypothalamic cDNA and genomic clones were isolated. PCR/Southernblot analysis of DNA isolated from human/rodent somatic cell hybridpanels was employed to determine the chromosomal localization of theCART gene. Northern blot analysis was also used to determine the grosspattern of distribution of CART mRNA in human brain. The high degree ofsimilarity at both the nucleic and amino acid level between rodent andhuman CART are suggestive of a conserved role within the mammalianneuroendocrine system.

Thus, the present invention relates generally to cocaine and amphetamineregulated transcript (CART) proteins and more particularly to mammalianCART proteins, to DNA sequences encoding CART proteins, to thepolypeptide products of recombinant expression of these DNA sequences,to peptides whose sequences are based upon the amino acid sequencesdeduced from these DNA sequences, to antibodies specific for suchproteins and peptides, to procedures for the detection and quantitationof such proteins and nucleic acids related thereto, as well as toprocedures relating to the development of therapeutic agents andpharmaceutical compositions utilizing CART proteins.

Association of DNA sequences provided by the invention with homologousor heterologous species expression control DNA sequences, such aspromoters, operators, regulators, and the like, allows for in vitrotranscription to form mRNA which, in turn, is susceptible to translationto provide CART proteins, and related poly- and oligopeptides in largequantities.

Also included within the invention is the incorporation of CART DNAsequences into procaryotic and eucaryotic host cells by standardtransformation and transfection processes, involving suitable viral andcircular DNA plasmid vectors, providing for useful proteins inquantities heretofore unavailable from natural sources.

In a presently preferred and illustrative DNA expression system of theinvention, CART protein encoding DNA in pBluescript SK⁻ is PCR amplifiedand digested with BamHI and XhoI for ligation into the plasmid pZVneo.The plasmid DNA is then cloned into vaccinia virus (Van Slyke et al.,"Use of Vaccinia Virus to Study Neuropeptide Processing," Methods inNeuroscience, Vol. 23: Peptidases and Neuropeptide Processing, A. I.Smith, Ed., Academic Press, San Diego, Calif. (1995)). The recombinantvaccinia virus, containing the CART protein encoding DNA, is used toinfect mammalian cells, such as HeLa, BSC40 (African Green Monkeykidney) or AtT-20 (mouse anterior pituitary corticotroph) cells allowingfor the production of a functional CART protein, demonstratingfunctional characteristics of native CART protein including for example,cross-reactivity with anti-serum to CART polypeptides.

In another representative DNA expression system of the invention, CARTprotein encoding DNA is ligated into bacterial plasmid pET15b or pET23bto obtain a pET/CART plasmid encoding a fusion protein comprising aminoacids encoded by the original pET plasmid, as well as the entire CARTsequence, minus the signal sequence. The pET/CART plasmid is thentransformed in Escherichia coli allowing for transcription andtranslation to provide a functional CART protein having a molecularweight of about 14 kD and/or about 17 kD demonstrating functionalcharacteristics of native CART proteins, including for example,cross-reactivity with anti-serum to CART polypeptides.

Novel protein products of the invention include polypeptides having theprimary structural conformation (i.e., amino acid sequence) of rat orhuman CART protein, such as that set forth in FIGS. 1A-1B and 2A-2B aswell as peptide fragments thereof, and synthetic peptides assembled tobe duplicative of amino acid sequences thereof. Proteins, proteinfragments, and synthetic peptides of the invention have numerous usesincluding therapeutic uses and provide the basis for preparation ofmonoclonal and polyclonal antibodies specifically immunoreactive withCART proteins. Antibodies of the invention can be used for affinitypurification of CART proteins from other sources and cell types, as wellas in the diagnostic quantification or qualification of CART proteins inhuman or animal biological samples.

The present invention also provides for procedures for the detectionand/or quantification of normal, abnormal, or mutated forms, of CARTproteins as well as nucleic acids (e.g., DNA and mRNA) associatedtherewith. In one illustrative embodiment, antibodies of the inventionare employed in known immunological procedures for quantitativedetection of CART proteins in samples, detection of DNA sequences of theinvention (particularly those having sequences encoding CART proteins)that may be suitably labelled and employed for quantitative detection ofmRNA encoding these proteins.

Among the multiple aspects of the present invention, therefore, is theprovision of novel purified and isolated DNA sequences coding forexpression of polypeptides having the characteristics of CART proteins(e.g., characterized by cross reactivity with antibodies to CART proteinor peptides) and including: (a) novel CART proteins encoded by DNAsequences set out in FIGS. 1 and 2, as well as (b) DNA sequences whichhybridize thereto under stringent hybridization conditions, i.e., of astringency equal to or greater than the conditions described herein andemployed in the initial isolation of DNAs of the invention, and (c) DNAsequences encoding the same, allelic variant, or analog CART protein orpolypeptide fragments, through use of, at least in part, degeneratecodons. Correspondingly provided are viral or circular plasmid DNAvectors incorporating such DNA sequences in procaryotic and eucaryotichost cells transformed or transfected with such DNA sequences andvectors, as well as novel methods for the recombinant production of CARTproteins through cultured growth of such hosts and isolation of theseproteins from the hosts or their culture media. Also, the CART proteinDNA can be used as a probe in the detection and isolation of variants ofCART proteins and analogs thereto. Diagnostic methods, therapeuticprocedures, and pharmaceutical compositions which utilize CART proteinsare also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other aspects and advantages of the invention will beapparent on consideration of the following detailed description and theaccompanying drawings, wherein:

FIG. 1A and 1B are a representation of the nucleotide sequence of ratCART cDNA (SEQ ID No.:3). The corresponding rat CART amino acid sequence(SEQ ID No.:4) is also shown. The poly(A) addition site (AATAAA)utilized to generate the approximately 700 base rat CART transcript isunderlined beginning at nucleotide 548. The poly(A) addition siteutilized to generate the approximately 900 base rat CART transcript isunderlined beginning at nucleotide 814. Open arrows represent the lastnucleotide prior to the site of addition of the two respective poly(A)tails. FIG. 1A and 1B also show the location at which the 5' PCRdifferential display oligonucleotide hybridized (nucleotides 158 through167) and the location at which the 3' PCR differential displayoligonucleotide hybridized (nucleotides 544 through 556), as isdescribed in Example 1. The translational reading frame begins with anATG at nucleotide 20 and extends to the TGA termination site atnucleotide 407. The region representing an alternately spliced 39nucleotide sequence is underlined at nucleotides 179 through 217. Thepresented nucleotide sequence has been assigned accession number U10071by GenBank);

FIG. 2A and 2B are a representation of the nucleotide and amino acidsequence of human CART cDNA (SEQ ID No.:7), as described in Example 4.The poly(A) addition site (AATAAA) utilized to generate theapproximately 900 base human CART transcript (nucleotides 780-785) isunderlined. The translational reading frame begins with an ATG atnucleotide 20, and extends to the TGA termination site at nucleotide368. The human CART cDNA sequence has been assigned accession numberU16826 by GenBank, with the human CART genomic DNA sequence assignedaccession number U20325;

FIG. 3A-3C are a representation of the nucleotide and amino acidsequence of human CART. For the nucleic acid sequence, capitalizedletters represent nucleotides present in both cDNA and genomic DNA,while lower case letters represent nucleotides found only in genomicDNA. A putative promoter element (tataaa) is underlined at position -31.Only partial intron sequences are shown, with the complete intron sizenoted in ˜bp, and the complete intron being set forth in SEQ ID No.:9. A39 base sequence which is alternately spliced in rat CART mRNA islocated at the 3' end of intron 1, and is italicized and underlined inFIG. 3A. The human CART mRNA cap site is located at nucleotide +1.Poly(A) addition sites (AATAAA) are underlined, with (A_(n))representing location of the poly(A) tail. The predicted translationalreading frame begins with an ATG at position +20 in the cDNA, andextends to the TGA termination site (*) at position 368. The human CARTgenomic DNA sequence has been assigned accession number U20325 byGenBank; and

FIG. 4 is a direct comparison of the rat CART protein amino acidsequence (designated "r-", SEQ ID No.:4) with the human CART proteinamino acid sequence (designated "h-", SEQ ID No.:8). Amino acidmismatches are shown by an asterisk (*). The underlined 13 amino acidsequence from amino acid 54 through amino acid 66 in the rat CARTprotein sequence is either present or absent due to an mRNA alternatesplicing event.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In one aspect, the present invention provides a method of recoveringCART protein in substantially pure form comprising the steps of removingthe supernatant from unlysed cells that express CART protein,introducing the supernatant to an affinity matrix containing immobilizedantibody capable of binding to CART protein, permitting the CART proteinto bind to the antibody of the matrix, washing the matrix to removeunbound contaminants, and recovering the CART protein in substantiallypure form by eluting said CART protein from said matrix.

The term "substantially pure" indicates a protein or composition that isessentially free of contaminants similar to the protein. In the presentcase, the normal contaminants associated with human CART proteinpredominately include human proteins. Thus, human CART protein issubstantially pure if it is essentially free of human proteins."Essentially free" is determined by weight. In general, a compositioncontaining 70% or more by weight human CART protein and less than 30% ofother human proteins may be considered substantially pure. Preferably,the composition will be at least 80% human CART protein, more preferablyat least 90%, and most preferably at least 95% human CART protein. Thepresence of dissimilar components does not affect the determination ofpurity, thus a composition containing 0.7 mg/mL human CART protein inPBS will still be considered substantially pure if it contains less than0.3 mg/mL other human proteins. In addition, further purificationutilizing a lectin or wheat germ agglutinin column may be used before orafter the antibody matrix step. Other purification steps could include,for example, sizing chromatography, ion chromatography, and gelelectrophoresis. Further purification by velocity sedimentation throughsucrose gradients may be used.

In another aspect of the invention, nucleotide sequences are providedthat encode CART protein, as well as the use of such sequences orfragments thereof in the production of recombinant CART protein, ashybridization probes, or for other purposes.

In another aspect, the invention further includes a method for producingan antibody which is capable of binding to CART protein or DNAcomprising the steps of preparing a peptide-protein ornucleotide-protein conjugate, said conjugate comprising at least 10,more preferably at least 14, and most preferably at least 18 consecutiveamino acid or nucleic acid residues present in CART protein or DNA,immunizing an animal with said peptide-protein or nucleotide-proteinconjugate, boosting the animals, and obtaining the antisera. Inconnection with this aspect, the present invention further includesmonoclonal and polyclonal antibodies specific for CART protein and DNA,i.e., capable of binding to a CART protein or nucleic acid molecule, aswell as hybridoma cell line capable of producing such an antibody.

In other aspects, the invention includes the use of antibodiesspecifically directed to CART protein or nucleotides, such as to isolateCART protein from sources producing the protein, for purposes ofdetermining the presence or amount of CART protein in a sample, and forother purposes apparent to those skilled in the art.

This invention further includes a method of diagnosis of the presenceand location of an CART protein expressing cell using labeled nucleotideprobe sequences or labeled antibodies of the invention.

In accordance with these and other aspects of the present invention,CART mRNA was initially identified using the differential display PCRtechnique of Liang et al., supra. In the differential display PCRtechniques, the general strategy is to amplify partial cDNA sequencesfrom subsets of mRNAs by reverse transcription and the polymerase chainreaction (PCR). These short sequences are then displayed on a sequencinggel. Pairs of primers are selected so that each will amplify DNA fromabout 50 to 100 mRNAs because this number is optimal for display on thegel.

Selection of 3' primers takes advantage of the polyadenylate poly(A)!tail present on most eukaryotic mRNAs to anchor the primer rate at the3' end of the mRNA, plus two additional 3' bases. A primer such as 5'T₁₁CA allows anchored annealing to mRNAs containing TG located justupstream of their poly(A) tails. By probability this primer willrecognize one-twelfth of the total mRNA population because there are 12different combinations of the last two 3' bases, omitting T as thepenultimate base. The primer permits initiation of reverse transcriptionof only this subpopulation.

Any reverse transcribed cDNA species is amplified by PCR if the distanceat which a second primer anneals is smaller than 2 to 3 kb from thebeginning of the poly(A) tail. Ideally this annealing position is within500 bp because cDNAs up to 500 bp can be resolved by size on a DNAsequencing gel. For a 5' primer of arbitrary base sequence, annealingpositions to cDNAs should be randomly distributed in distance frompoly(A) tail. Therefore, the amplified products from various mRNAs willdiffer in size. After these PCR products have been labeled with α-³⁵S!-labeled deoxyadenosine triphosphate (dATP), they are displayed byautoradiography as a ladder on a sequencing gel.

The 5' primer should in theory be short, 9 to 11 bp, for it to annealfairly frequently within a cDNA strand. To permit such short primers togive specific DNA amplification by PCR, PCR parameters are chosen thatare optimal for product yield and specificity, such as 40° C. annealing(to allow for some mismatch to occur) and a 30 sec elongation time thatallows amplification of short products that can be resolved by a DNAsequencing gel.

PCR generated cDNA representing an mRNA is then recovered from a driedDNA sequencing gel and reamplified with PCR. A DNA band from thesequencing gel is eluted and precipitated to remove contaminants such asurea. The recovered DNA is reamplified in the presence of 20 μM dNTP.The subsequent reamplified PCR DNA is then subcloned and used as a probefor further analysis.

To determine the usefulness of PCR differential display as an approachby which to identify cocaine and amphetamine regulated transcripts, theefficacy of the technique was evaluated by utilizing c-fos specificoligonucleotides capable of generating a PCR product from the rat c-fostranscript (Curran et al., "Isolation and Characterization of thec-fos(rat) cDNA and Analysis of Post-translational Modification invitro," Oncogene, 2:79-84 (1987)). The observed profile of radiolabeledPCR products showed, as expected, that multiple species of mRNA withinthe rat cerebellum, striatum and hippocampus can serve as ahybridization target for the two oligonucleotides utilized. A typicalreaction generated 50-200 distinct radiolabeled PCR products between 50and 600 bases in length. Furthermore, the overwhelming majority of PCRproducts were present at identical levels in these three brain regionsisolated from saline, cocaine and amphetamine treated animals. The PCRproduct representing the c-fos transcript was, however, clearly inducedin the striatum and cerebellum from cocaine and amphetamine treatedanimals, with no such induction observed in the hippocampus. Therelative levels of the c-fos PCR products were determined bysemi-quantitative densitometric analysis, and the degree of inductionwas nearly identical to that determined previously by Northern blotanalysis. Thus, not only is PCR differential display capable ofdetecting qualitative differences in relative mRNA levels, but issemi-quantitative as well under the PCR conditions employed.

PCR differential display allows for the microanalysis of transcriptionalchanges occurring in a given cell or tissue (Liang and Pardee, supra;Liang et al., supra; Bauer et al., supra). In accordance with thepresent invention, brain (striatum, cerebellum and hippocampus)transcripts were identified whose relative levels are regulatedfollowing acute administration of the psychomotor stimulants, cocaineand amphetamine. 96 individual PCR differential display reactions wereperformed using 12 different 3' primers and 8 different 5' primers. Over12,000 PCR products were generated, and less than 0.05% of the observedbands exhibited profiles indicative of transcriptional regulation bycocaine or amphetamine. Thus, acute psychomotor stimulant administrationappears to affect transcription of an extremely limited array of geneswithin the brain structures examined, and clearly does not inducewholesale transcriptional effects within the 60 minute time frameexamined.

One PCR product from rat brain was identified which represents an mRNAdoublet whose relative striatal levels are increased 4- to 5-fold byboth acute cocaine and amphetamine treatment. This observation led tothe investigation of the effects of another addictive drug, morphine, ontranscriptional expression in various rat brain regions. Acute morphine(10 mg/kg, with animals sacrificed 60 minutes following injection) hadno apparent effect on RNA doublet levels within the striatum, cortex,hypothalamus, hindbrain, midbrain or hippocampus. Chronic morphineadministration (one 75 mg morphine pellet implanted daily for 7 days),and naloxone-precipitated morphine withdrawal (60 minutes post 0.1, 1and 100 mg/kg naloxone administration) also had no observable effect onRNA doublet levels. Thus, modulation of the catecholaminergic systemappears to control RNA doublet levels in the striatum only, withmodulation of the opioid system having no observable effect on neuronaltranscription.

cDNA sequence analysis characterized the rat mRNA corresponding to thestriatal, psychomotor stimulant regulated PCR product. The complete ratcDNA is shown as SEQ ID No.:3. At least two distinct transcriptionalevents occur in a differential fashion to generate multiple species ofmRNA from a unique primary transcript. One such event involvesdifferential poly(A) site utilization. The poly(A) addition eventpresumably occurs approximately equally at the two AATAAA sites noted inFIG. 1B, as transcriptionally active tissues contain nearly equivalentlevels of the 700 and 900 base transcripts. As shown in FIG. 2A-2B thecorresponding human transcript is present as a single 900 base species,suggesting that the upstream poly(A) addition site is either not presentor not utilized within the primary human transcript. Differential exonsplicing in the rat transcript represents a second type oftranscriptional event producing multiple species of mRNA. Unlikedifferential poly(A) site utilization, this event is predicted to effectthe ultimate translation product, as the alternately spliced 39 basesequence element is located within the putative protein coding region.Approximately two thirds (11 out of 16) of the rat cDNA clones examinedwhich span the region did not contain the 39 base pair sequence, whereasapproximately one third (5 of 16) of the rat cDNA clones examined whichspan this region contained the 39 bp insert. The rat CART DNA which doesnot contain the 39 bp insert is hereinafter referred to as rCART1 DNA,whereas the rat CART DNA containing the 39 bp insert is hereinafterreferred to as rCART2 DNA. Also, Northern blot analysis of rat midbrainand hypothalamic total RNA using a radiolabeled oligonucleotidecomplementary to the 39 bp insert showed that the sequence element ispresent in both the 700 and 900 base transcripts in both tissues. Thus,nuclear factors which determine apparent alternate splicing do notappear to be coupled to events determining poly(A) site utilization.

Northern blot analysis identified the rat brain regions and peripheraltissues which express the RNA doublet. A limited profile of expressionwas observed, with the hypothalamus and pituitary representing the majorsites of transcriptional expression. The midbrain/thalamus and eye alsocontained relatively abundant levels of the RNA doublet. Other brainregions, as well as the adrenal, contained small yet detectable levelsof the transcript. This pattern of expression is suggestive of apotential functional role of the predicted protein product within theneuroendocrine system. Expression within human brain was qualitativelysimilar to that observed for rat, indicating that the protein productalso plays a functional role across mammalian species. Lastly, the useof both sense and antisense strand-specific RNA probes in Northern blotanalysis revealed that only one strand of the corresponding gene istranscribed; no hybridization signals were observed in any RNA sampletested when the sense RNA was used as a hybridization probe.

Distribution of the RNA doublet throughout the rat brain as shown by insitu hybridization was predominantly confined to hypothalamicneuroendocrine neurons and limbic neural circuits. Contributions fromstructures in sexual circuits and those involved in autonomic functionsalso exist. This unique brain distribution may provide some insightsinto a possible functional role for the encoded protein product. Forexample, expression of the RNA doublet is robust in the hypothalamus.Its abundance in this brain region is due, in part, to expression withinneuroendocrine neurons such as the paraventricular, arcuate andsupraoptic nuclei. Many neurons within these regions send afferents to,and release neuropeptides which modulate pituitary hormone secretion andregulate osmolality (Simerly, "Anatomical Substrates of HypothalamicIntegration," In The Rat Nervous System, (Paxinos, Ed.), in press,Academic, New York, N.Y. (1994)). Thus, localization of high levels ofthe RNA doublet to these neurons, coupled with the predicted structureof the encoded protein product(s) suggest that the transcript may encodea neuroendocrine secretogogue.

Another possible role for the encoded protein is suggested by expressionof the RNA doublet in rat limbic neural circuits. For example, thetranscript is present within the amygdaloid complex, dentate gyrus ofthe hippocampus, and the hypothalamic mammillary nucleus andsupramammillary nucleus. Expression within the neostriatum is unique byits confinement to the nucleus accumbens and relative absence from thecaudoputamen. The nucleus accumbens mediates the reinforcing andrewarding properties of drugs of abuse (Koob, "Drugs of Abuse: Anatomy,Pharmacology and Function of Reward Pathways," Trends Neurosci.,13:177-184 (1992)). Furthermore, the striatum is the only brain regionthat exhibited up-regulation of rat RNA doublet levels following acutecocaine and amphetamine administration. Such a limited pattern ofexpression throughout neostriatal, hypothalamic and amygdaloidcomponents of the limbic neural circuits suggest a possible role inreward processes and affect.

The remaining areas of the rat brain exhibiting RNA doublet expressionare limited, yet may provide additional information regarding functionof the protein. For example, moderate levels of expression are seen intwo nuclei which represent major sexual neural circuits - the ventralpremammillary nucleus of the hypothalamus, and the posterior dorsalaspect of the medial nucleus of the amygdala (which receives afferentsfrom the former structure). The ventral premammillary nucleus receivesinputs from sexually dimorphic brain regions and has been implicated inmediating reproductive behavior and physiology as well as aggressivebehavior (Simerly, supra). Rat RNA doublet expression is also observedwithin sites that modulate autonomic functions. Neurons of the nucleussolitary tract and inferior olive contain moderate to weak levels of theRNA doublet. Noradrenergic neurons of the brainstem such as the locuscoeruleus and cells within the Al/Cl region of the ventrolateral medullaexpress the transcript. The locus coeruleus projects extensivelythroughout the brain and spinal cord and participates in arousal states(Aston-Jones et al., "Anatomy and Physiology of the Locus CoeruleusNeurons: Functional Implications," In Norepinephrine: Frontiers inClinical Science (Ziegler and Lake, Eds.), Williams and Wilkins,Baltimore, Md., pp. 92-116 (1984)).

As shown in FIG. 1A-1B rat cDNA sequence analysis predicts thetranslation of a protein either 129 (SEQ ID No.:4) or 116 (SEQ ID No.:6)amino acids in length, depending on the presence or absence of the 39base in-frame sequence element. Furthermore, polysome analysis indicatesthat the RNA doublet is efficiently and actively translated in thehypothalamus in vivo, with in vitro translation resulting in thegeneration of protein products whose observed apparent molecular masscorrelates with those predicted solely from nucleotide sequenceanalysis. Additional computer analysis (employing the BLAST alignmentprogram; Altschul et al., "Basic Local Alignment Search Tool," J Mol.Biol., 215:403-410 (1990)) of the predicted proteins against the 113,000protein sequences contained within the NIH data base revealed nosignificant homology. The most noteworthy property of the encodedproteins is the presence of a hydrophobic amino terminal domain.Nineteen of the initial 27 residues are hydrophobic, representing theonly region of the predicted protein constituting a major hydrophobicdomain. Furthermore, the overall structure of this region corresponds tothat of a signal peptide motif (Lingappa and Blobel, "Early Events inthe Biosynthesis of Secretory and Membrane Proteins: the SignalHypothesis," Rec. Prog. Hormone Res., 36:451-475 (1980); van Heijne,"The Signal Peptide," J Membr. Biol., 115:195-201 (1990)), and suggeststhat the protein products are targeted for secretion followingsynthesis. Another potentially significant observation is the presenceof multiple paired basic amino acid residues (RR, KR, RK and KK) locatedthroughout the predicted protein sequence. Such sites represent thoserecognized by various neuroendocrine protein convertases (Steiner etal., "The New Enzymology of Precursor Processing Endoproteases," J Biol.Chem., 267:23435-23438 (1992)). Thus, the possibility arises that thepredicted protein may be a substrate target for post-translationproteolytic cleavage events.

A human hypothalamic cDNA and genomic DNA library were screened with aradiolabeled full-length rat CART cDNA probe under stringent conditionsto isolate human CART cDNA and genomic clones. The full length humancDNA sequence (SEQ ID No.:7) is approximately 800 bases in length,excluding the poly(A) tail which was present on several cDNA clones.Although three poly(A) addition recognition sites (AATAAA) are presentwithin the 3' untranslated region, cDNA clone analysis coupled withNorthern blot studies indicate that only one is predominantly utilizedin vivo. This is in contrast to the rat CART transcript, where twopoly(A) sites are recognized at an apparently equal frequency in vivo,resulting in the presence of a distinct RNA doublet following Northernanalysis (Douglass et al., "PCR Differential Display Identifies a RatBrain mRNA That is Transcriptionally Regulated by Cocaine andAmphetamine," J Neurosci., 15:2471-2481 (1995). Nucleotide sequencecomparison to full length rat CART cDNA reveals an overall identity ofapproximately 80%, with a 92% degree of homology observed within thepredicted protein coding region. The majority of the latter differencesare at third base codon locations.

Comparison of the human cDNA sequence (SEQ ID No.:7) to that of thegenomic DNA (SEQ ID No.:9) reveals that mature human CART mRNA isencoded by three distinct exons. The two resulting introns arerelatively small (approximately 425 and 540 bp in length) and arelocated within the presumed CART protein coding region. In the 5' geneflanking region a consensus TATAAA box is observed at position -31,indicative of a functional promoter element, while in the 3' flankingregion the genomic DNA sequence diverges from the cDNA sequence at thesite of poly(A) addition, as expected.

Analysis of human CART genomic DNA also reveals the apparent molecularbasis for an alternate splicing event which occurs in the rat CARTtranscript (see FIG. 3A-3C). In the rat, a 39 base sequence is presentin approximately one-third of the mature CART transcripts expressed inthe striatum and hypothalamus as determined by both cDNA sequence andPCR analysis. This sequence, when present, is found within the predictedCART protein coding region, resulting in the presence or absence of a 13amino acid sequence within the CART protein. Analysis of human CARTgenomic DNA identifies a highly related 39 base sequence at the 3' endof the first intron (FIG. 3A). 25 of 39 nucleotides are conservedbetween the two species, with an even lower degree of conservation (6 of13) observed at the corresponding amino acid level. It is currentlyunclear as to why this 39 base sequence is alternately spliced in ratbrain tissues expressing the CART transcript, while no such event isobserved following an identical analysis of human hypothalamic RNA.

The first potential translation initiation codon is located atnucleotide 20 within human CART cDNA, and the cDNA coding sequenceextends to the TGA termination site (*) at nucleotide 368, predictingthe generation of a protein 116 amino acid residues in length. The humanCART mRNA cap site is located at nucleotide +1 (FIG. 3A). The mostnoteworthy property of the predicted protein is the presence of an aminoterminal hydrophobic domain (27 residues in length) conforming to themotif of a signal peptide (Lingappa and Blobel, supra; von Heijne etal., supra) and suggesting that the protein is targeted for entry intothe secretory pathway. The sequence of the predicted human CART proteinis 95% identical to that for rCART1 (see FIG. 4). Within the hydrophobicamino terminal domain, three conservative (V6L, L11V, and M19L) and twononconservative (T25A, and R26G) substitutions are observed. However,throughout the remaining 90 residues only one conservative substitution(V691) is present. This observation of significant conservation at theamino acid sequence level is consistent with the notion that the CARTprotein plays a conserved role within the mammalian neuroendocrinesystem.

The use of hCART DNA and rCART DNA, or fragments thereof, as a probe inthe isolation, purification, and study of other CART proteins from otherorganisms is contemplated. Oligonucleotide fragments of CART DNA canalso be used as primers to amplify (with specific DNA polymerases)genomic DNA, isolated, for example, from bacteria, fungi, avian, andmammalian sources. The amplified genomic DNA can then be analyzed toidentify sequence variation/abnormality using the polymerase chainreaction assay (Saiki et al., Science 230:1350 (1985). See also, Mullis,K. B., U.S. Pat. No. 4,683,202, Jul. 28, 1987; and Mullis, K. B., U.S.Pat. No. 4,683,195, Jul. 28, 1987).

For the analysis of mRNA for CART, or mRNA for related proteins, dothybridization and Northern hybridization analyses can be used tocharacterize mRNA encoding CART protein or CART protein-like moleculesquantitatively and qualitatively. From these studies valuableinformation can be obtained about the number of different forms of CARTgenes and their expression in various cell types, e.g., bacteria, fungi,arian, and mammalian.

Thus, in addition to characterization of CART DNA and polypeptidesdescribed in detail above, the present invention has the followingadvantages:

(1) The nucleic acids coding for rCART or hCART can be used as probes toisolate other members of the CART gene family.

(2) The nucleic acids coding for rCART or hCART can be used to deriveoligonucleotide probes to determine the presence of CART mRNA and theexpression of CART and other CART genes in various tissue types.

(3) rCART or hCART nucleotide sequences can be used to predict theprimary amino acid sequence of the protein for production of syntheticpeptides.

(4) Synthetic peptides derived from the above sequences can be used toproduce sequence-specific antibodies and antibody fragments.

(5) Immunoassays for CART polypeptides can be produced with thesesequence-specific antibodies and synthetic peptides.

(6) The aforementioned immunoassays can be used as diagnostics forcocaine or amphetamine usage, and/or for susceptibility orpredisposition to cocaine or amphetamine addiction.

Peptides according to the present invention can be labelled byconventional means using radioactive moieties (e.g., ¹²⁵¹ I), enzymes,dyes, fluorescent compounds, and other conventional labels. Severalpossible configurations for immunoassays according to the presentinvention can be used. The readout systems capable of being employed inthese assays are numerous and non-limiting examples of such systemsinclude fluorescent and colorimetric enzyme systems, radioisotopiclabelling and detection and chemiluminescent systems. Two illustrativeexamples of immunoassay methods are as follows:

(1) An enzyme linked immunoassay (ELISA) using an antibody preparationaccording to the present invention (including Fab or F(ab)' fragmentsderived therefrom). To a solid phase (such as a microtiter plate orlatex beads) is attached a purified antibody or antibody fragment havinga specificity for CART polypeptides. This solid phase antibody iscontacted with the sample containing CART. After washing, the solidphase antibody-antigen complex is contacted with an enzyme-conjugatedanti-peptide antibody (or conjugated fragment) having a CART bindingspecificity different from that of the solid phase antibody. Afterwashing away unbound conjugate, color or fluorescence is developed byadding a chromogenic or fluorogenic substrate for the enzyme. The amountof color or fluorescence developed is proportional to the amount of CARTin the sample.

(2) A competitive fluorometric immunoassay using fluorescently labelledpeptide or synthetic peptides of the sequences for CART polypeptides. Inthis example, the purified peptide expressed by cells or syntheticpeptides thereof are fluorescently labelled. To a solid phase isattached a purified antibody having a binding specificity for CARTpolypeptides. This solid phase is then contacted with sample containingCART polypeptide to which has been added fluorescent peptide probe.After binding, excess probe is washed away and the amount of bound probeis quantitated. The amount of bound fluorescent probe will be inverselyproportional to the amount of CART polypeptide in the sample.

In the nucleic acid hybridization method according to the presentinvention, the nucleic acid probe is conjugated with a label, forexample, an enzyme, a florophore, a radioisotope, a chemiluminescentcompound, etc. In the most general case, the probe would be contactedwith the sample and the presence of any hybridizable nucleic acidsequence would be detected by developing in the presence of achromogenic enzyme substrate, detection of the fluorophore byepifluorescence, by autoradiography of the radioisotopically labelledprobe or by chemiluminescence. The detection of hybridizable RNAsequences can be accomplished by (1) a dot blot methodology or (2) an insitu hybridization methodology. Methods of these last two techniques aredescribed by Gillespie and Bresser, "mRNA Immobilization in NaI: QuickBlots," Biotechniques, 184-192, (November/December 1983) and Lawrenceand Singer, "Intracellular Localization of Messenger RNAs for CytosketalProteins," Cell 45:407-415 (1986), respectively. The readout systems canbe the same as described above, e.g., enzyme labelling, radiolabelling,etc.

The foregoing and other aspects of the invention may be betterunderstood in connection with the following examples, which arepresented for purposes of illustration and not by way of limitation.

EXAMPLES Example 1 Differential Display of Rat CART-RNA

Adult 90-day old male Sprague-Dawley rats were kept under a 12 hrlight/12 hr dark cycle and given food and water ad libitum. A singledose of cocaine (20 mg/kg) dissolved in saline, amphetamine (6 mg/kg)dissolved in saline or saline alone (as control) was givenintraperitoneal (i.p.) to each of 6-10 rats. The animals were sacrificed1 hr after injection via halothane anesthetization to unconsciousness(<1 min) and decapitation. The whole brain was removed and rinsed inice-cold PBS for 1 minute prior to dissection. Cerebellum, striatum andhippocampus were dissected and immediately stored on dry ice at -70° C.

Brain dissections were performed as described (Glowinski and Iversen,"Regional Studies of Catecholamine in the Rat Brain: The Disposition of³ H!norepinephrine, ³ H!dopamine, and ³ H!DOPA in Various Regions of theBrain," J Neurochem., 13:665-669 (1966)). Total RNA from theaforementioned brain regions (plus additional brain regions) andperipheral tissues was immediately prepared using a modified acid phenoltechnique (Chomczynski and Sacchi, "Single-Step Method of RNA Isolationby Acid Quanidinium Thiocyanate-Phenol-Chloroform Extraction," Anal.Biochem., 162:156-159 (1987)), with final concentration determinedspectrophotometrically.

Following the determination of concentration, RNAs from the cerebellum,striatum and hippocampus were pooled from 6 to 10 animals receivingeither the saline, cocaine or amphetamine injections. Reversetranscription was performed essentially as described previously (Liangand Pardee, supra) using T₁₁ VN oligonucleotides as primers. The onlysignificant modification involved a primer pre-annealing step at 35° C.for 60 minutes prior to initiation of reverse transcription by theaddition of dNTPs and MMLV reverse transcriptase. Reverse transcriptionreactions were then subjected to PCR in the presence of ³⁵ S-α-dATP andoligonucleotides specifically designed for differential display (Liangand Pardee, supra). Final concentrations of 1 mM MgCl₂ and 20 μM dNTPswere found to be optimal for these two reagents. The 5' PCRoligonucleotide (10 mer, 50% G/C content, ACGTCTCATG, SEQ ID No.:1) waspresent at a final concentration of 0.5 pmol/μl, while the 3' PCRoligonucleotide (TTTTTTTTTTTGC, SEQ ID No.:2) was present at a finalconcentration of 2.4 pmol/μl. PCR conditions employing a Perkin ElmerCetus GeneAmp PCR System 9600 thermocycler were: 40 cycles ofdenaturation at 94° C. for 30 sec, primer annealing at 40° C. for 120sec, and extension at 72° C. for 30 sec. A final extension reaction wasthen performed at 72° C. for 7 min. Radiolabeled reaction products weresubjected to high resolution polyacrylamide/urea gel electrophoresis asdescribed (Liang and Pardee, supra). The relative abundance of anapproximately 400 bp PCR product was significantly increased in striatalRNA samples from animals treated acutely with cocaine or amphetamine. Nosuch drug induced increase was observed for the hippocampal RNA samples,and no PCR product was observed in the cerebellar RNA samples regardlessof drug treatment. To further characterize this PCR product, thefragment was isolated from a preparative acrylamide/urea gel, subjectedto PCR under standard conditions, and subcloned into the pCRII vector(Invitrogen, San Diego, Calif.) via the T/A cloning procedure. Insertswere analyzed by supercoil dideoxynucleotide sequencing.

To confirm that the cloned PCR product represents a striatal mRNA whichis transcriptionally regulated by psychomotor stimulants, the purifiedDNA fragment was radiolabelled to serve as a hybridization probe forNorthern blot analysis. For Northern blot analysis, total RNA (3-5 μg)was separated by electrophoresis on 6% formaldehyde/1.2% agarose gels.The RNA was transferred to Magna NT nylon membranes (Micron SeparationsInc., Westboro, Mass.) by capillary action with 20×SSC, followed by UVcross-linking. The membranes were then briefly dipped in 0.3 M sodiumacetate, 0.02% methylene blue to stain the transferred RNA. 28S and 18SrRNA were readily apparent, and served as a means by which tostandardize for the amount of RNA contained within each sample.Following removal of stain by boiling in water for 10 min, the membraneswere prehybridized for 3-24 hr at 60° C. in hybridization buffer (5%SDS, 400 mM sodium phosphate (7.0), 1 mM EDTA, 1 mg/mil BSA (fraction V)and 50% formamide). The prehybridization buffer was discarded andreplaced with fresh hybridization buffer including the hybridizationprobe. Hybridization proceeded for 16-24 hr at 42° C. Membranes weresubsequently washed with 1% SDS, 0.5×SSC and 1 mM EDTA at 50°-55° C. for1-4 hr. After washing, membranes were exposed multiple times to KodakXAR-5 film in order to obtain a range of hybridization signalintensities for semi-quantitative densitometric analysis. For each RNAsample, autoradiographic signals within the linear range of filmsensitivity were digitized using an X-Ray Scanner Corp. model MSF300ZSlaser scanner and Adobe Photoshop XSF software. Relative intensitieswere quantified using Image software. RNA doublet signal intensitieswere standardized against 28S or 18S rRNA (detected by methylene bluestaining), or cyclophilin mRNA (detected by Northern blot analysisemploying radiolabeled cRNA as a hybridization probe). In striatal andhippocampal RNA isolated from saline injected animals, two hybridizationpositive transcripts were observed which are approximately 700 and 900bases in length. In the striatum, cocaine and amphetamine treatmentincreased the relative level of the RNA doublet by 4- to 5-fold, whileno such modulation was observed in the hippocampus. Additional Northernblot analysis of RNA isolated from hypothalamus, midbrain/thalamus,hindbrain and cortex also showed no transcriptional regulation by acutecocaine or amphetamine treatment. No RNA doublet was observed incerebellar RNA samples regardless of treatment. Thus, the profile ofthis RNA within the cerebellum, striatum and hippocampus as determinedby Northern blot analysis conforms to that for the corresponding PCRproduct identified by differential display analysis.

Example 2 Isolation and Cloning of Rat CART cDNA

Following confirmation that the PCR product from Example 1 was derivedfrom a psychomotor stimulant regulated transcript, rat striatal andhypothalamic cDNA libraries were screened under stringent hybridizationconditions utilizing the radiolabelled PCR fragment as a hybridizationprobe. Specifically, cDNA clones were isolated from rat striatal andhypothalamic libraries in the lambda vector, ZAPII (libraries purchasedfrom Stratagene, La Jolla, Calif.). Approximately 5×10⁵ clones wereplated from each of the aforementioned libraries, and screened understringent hybridization conditions employing the radiolabeled PCRfragment as a hybridization probe. About 20 distinct hybridizationpositive clones were plaque purified, the cDNA inserts isolated inphagemid form following superinfection with helper phage, and theinserts sequenced as described above. Subsequent nucleotide sequenceanalysis was performed employing the Wisconsin CGC suite of analyticalsoftware. The nucleotide sequence of the full length cDNA has beenassigned accession number U10071 in the GenBank database. 5' RACE wasalso used to isolate cDNA/PCR products representing the 5' end of themRNA. The rat cDNA sequence (SEQ ID No.:3) representing theapproximately 900 base transcript is shown in FIG. 1A-1B. The cDNA is840 bases in length, excluding an (A)₂₆ tail. The poly(A) additionrecognition sequence, AATAAA, at position 814 directs poly(A) tailaddition at this site. A second species of cDNA clone contained an (A)₄₀tail following nucleotide 579, utilizing the AATAAA element at position548, to produce an approximately 700 base transcript. Thus, themolecular mechanism underlying the appearance of the RNA doublet appearsto be differential poly(A) site utilization. cDNA nucleotide sequenceanalysis also uncovered an apparent alternate exon splicing event withinthe predicted translated region of the RNA. Approximately one third ofthe cDNA clones sequenced contained the 39 nucleotides representingthose from 179-217, while the remaining clones did not. Lastly,nucleotides 158 and 556 represent the 5' ends of the twooligonucleotides used in the original PCR differential display analysis.Several mismatches are present between the PCR differential displayoligonucleotides and the complementary cDNA sequence, as anticipated.The predicted translational reading frame begins with an ATG atnucleotide 20 and extends to the TGA termination site at nucleotide 407.An arrow in FIG. 1B denotes the carboxyterminal residue of the predictedhydrophobic signal sequence. The sequence of the original PCRdifferential display fragment also matches that located betweennucleotides 168 and 543 in the cDNA clones.

As described above, the first potential translation initiation codon islocated at nucleotide 20. In 90-95% of the vertebrate transcriptsanalyzed, the first 5' AUG represents the site for translationinitiation (Kozak, "The Scanning Model for Translation: An Update," J.Cell Biol., 108:229-241 (1989)). Furthermore, the nucleotide sequence inwhich this ATG is embedded is similar to that representing a consensusKozak translation initiation sequence element (GCCGCCRCCATGG) (SEQ IDNO.:14). The predicted encoded polypeptide is either 129 or 116 residuesin length, depending on the presence or absence of nucleotides 179-217within the putative protein coding region, respectively. The first 27residues represent an amino terminal hydrophobic domain conforming tothe motif of a signal peptide (Lingappa and Blobel, supra; van Heijne,supra), suggesting that the polypeptide may be targeted for entry intothe secretory pathway. There are also multiple paired basic amino acidresidues located throughout the molecule which could theoretically serveas target sites for post-translation proteolytic cleavage events(Steiner et al., supra). One such site is present within the alternatelyspliced domain encoding residues 54-66, further suggesting that ifproteolytic cleavage events did occur, then different peptide endproducts would be produced from the two distinct primary translationproducts.

Example 3 Distribution of Rat CART RNA Within the Adult Rat Brain andPeripheral Tissues

For in situ hybridization analysis of rat brain, a slightly modifiedversion of the Simmons et al., "A Complete Protocol for in situHybridization of Messenger RNAs in Brain and Other Tissues WithRadiolabelled Single-Stranded RNA Probes," J Histotechnol, 12:169-181(1989) protocol was used. Serial sections 30 μm thick from three adultmale Sprague-Dawley rat brains were cut on a sliding microtome. Wholebrain series were collected from prefrontal cortex to the cervicalspinal cord, and each section in a series was approximately 270 μm froman adjacent section. Free floating sections were stored incryoprotectant (50% 0.05 M sodium phosphate buffer, pH 7.3, 30% ethyleneglycol, 20% glycerol) at -20° C. for 2-6 weeks. Sections were thenmounted onto subbed and poly-L-lysine coated slides after 3 washes (in0.05 M sodium phosphate buffer, pH 7.3), vacuum dried, and storeddesiccated at -70° C. until hybridization. Hybridization with a nearfull length rCART ³⁵ S-α-UTP radiolabeled antisense cRNA probe wasperformed at 63° C., with the most stringent post-hybridization washstep performed at 75° C. Hybridization with a sense RNA probe served asa control for in situ signal specificity. Slides were exposed to CRONEXfilm (Amersham) for 2 days. They were then dipped in NB-20 emulsion(Kodak) and developed 5 days thereafter.

To identify specific neuroanatomical structures, adjacent sections wereNissl stained followed by microscopic evaluation. The rat brain atlas ofSwanson, "Brain Maps: Structure of the Rat Brain," Elsevier, New York,N.Y. (1992), was used to identify specific in situ hybridizationpositive cells and brain nuclei, as well as providing a nomenclature forsubsequent labeling.

Analysis of relative rCART RNA levels within gross brain structures andperipheral tissues was carried out by Northern blot analysis with anantisense cRNA probe following subcloning of the rCART cDNA fragmentrepresenting nucleotides 42-800 into the plasmid pBSII SK- (Stratagene).A 650 bp fragment (35-685, PstI/HincIl) of rat cyclophilin cDNA was alsosubcloned into pGEM 3Z, with relative cyclophilin mRNA levels serving asinternal controls. Radiolabelled (³² P-α-UTP) RNA probes weresynthesized in vitro using either SP6, T3 or T7 RNA polymerase. Specificactivities of cRNA probes were routinely greater than 3×10⁹ cpm/μgplasmid.

The major site of synthesis of the CART RNA is the hypothalamus.Relatively abundant RNA levels were also observed in thethalamus/midbrain, with significantly lower expression observed in thecortex, striatum, hippocampus and hindbrain. Semi-quantitativedensitometric analysis of the hybridization signals revealed thathypothalamic CART RNA levels are approximately 300-fold greater, andmidbrain/thamus RNA levels 10-fold greater than striatal levels.Analysis of RNA isolated from peripheral tissues also detected thepresence of the CART RNA in rat eye and adrenal, while no hybridizationsignal was observed in the other 13 tissues examined. Levels of the CARTRNA in the eye approximated those of the midbrain/thalamus, whileadrenal levels are similar to those observed in the striatum. Severaladditional endocrine tissues were also evaluated for expression of theCART mRNA. No hybridization signal was observed in rat ovary and uterusRNA. However, a robust hybridization signal was observed when pituitaryRNA was examined; the relative intensity of the pituitary hybridizationsignal approximates that observed for the hypothalamus. Expression ofthe CART RNA is thus limited to neuroendocrine tissues, withdramatically varied levels of transcriptional expression observedthroughout the brain.

Further Northern blot analysis also confirmed that the CART transcriptis present in RNA isolated from human brain. Relatively abundant levelsof an approximately 900 base hybridizing transcript were observed in RNAfrom human hypothalamus, frontal cortex and midbrain, with lower levelsseen in the hippocampus, motor cortex and striatum. No suchhybridization signal was observed in cerebellar RNA. Thus, the patternof distribution of the transcript in human brain was relativelyconserved to that observed for rat brain. However, only a single speciesof hybridizing RNA was present in human, in contrast to the doublet RNAband which was consistently observed in rat.

Regional expression of the CART RNA throughout the adult rat brain wasmore precisely determined by in situ histochemical analysis. For thesestudies a 790 base cRNA hybridization probe spanning the putative codingregion (including the alternately spliced 39 base insert) and 3'noncoding region was utilized. The most intense hybridization signalswere observed in the Edinger-Westphal nucleus and induseum griseum.Major expression was also seen in the periventricular zone of thehypothalamus (especially the paraventricular and arcuate nuclei) andcells throughout the medial hypothalamus (typified by labeling of thesupraoptic nucleus and peri-fornical region). Within these structures,intense hybridization signals were observed at the single cell level.

Telencephalic hybridization signals were limited to a few areas. Of notewas the distribution in the neostriatum where moderate labeling wasevident in the ventral region, consisting of the nucleus accumbens andolfactory tubercle. This area of hybridization positive cells stretchedalong the nucleus accumbens border within striosomes of theventrolateral caudoputamen. Within the neocortex, moderate labeling wasseen only in the primary somatosensory area (layer 4) and the piriformarea. Intense labeling was observed in the induseum griseum, with thebed nuclei of the stria terminalis and the dorsal blade of the dentategyrus of the rostral hippocampus exhibiting moderate to weak labeling.Moderately intense hybridization signals were also seen in theamygdaloid complex, particularly in the medial part of the septalnucleus, posterior dorsal part of the medial nucleus, posterior corticalnucleus, and posterior basolateral nucleus.

In the diencephalon, the hypothalamus exhibited the most extensivelabeling distribution of any brain region examined. Intense to moderatelabeling of cells was evident throughout entire nuclei such as theparaventricular (particularly the parvocellular region), arcuate andsupraoptic nuclei. Moderate labeling was seen in the posteriorperiventricular nucleus, ventral and medial premammillary nucleus, andlateral aspect of the supermammillary nucleus. Scattered cells in theperi-fornical region, lateral hypothalamus, and posterior hypothalamusexhibited intense to moderate signals. Within the thalamus, the medialaspect of the zona incerta, ventral half of nucleus reuniens, andlateral habenula were the only structures exhibiting moderate to weaklyintense hybridization signals.

The mesencephalon contained the fewest number of hybridization positivecells. However, within this region the Edinger-Westphal nucleusexhibited intense labeling. Scattered cells with moderate to weaklabeling were also noted in the dorsal periaqueductal gray. Therhombencephalon also contained regions of hybridization positive cells.Both the inferior olive and nucleus of the solitary tract exhibitedmoderate labeling, where as the locus coeruleus and nucleus incertusshowed weak labeling. Scattered cells throughout the Al/Cl region of thenucleus ambiguus further exhibited moderate labeling.

Hybridization of all aforementioned brain sections with a sense CARTcRNA probe under identical conditions showed no labeling of any of thestructures described above. Thus, the hybridization signals which wereobserved employing the antisense cRNA probe were specific, andrepresented cells expressing the CART RNA.

Example 4 Isolation and Characterization of Human CART cDNA and GenomicDNA

A human hypothalamic cDNA and genomic DNA library were screened with aradiolabeled, full-length rat CART cDNA probe under stringenthybridization conditions to isolate human CART cDNA and genomic DNAclones. cDNA clones were isolated from a human hypothalamic cDNA libraryin the lambda vector ZAPII (library #77425, obtained from ATCC,Rockville, Md.). Genomic clones were isolated from a human genomic DNAlibrary in the lambda vector Charon 4A (library #37385, also obtainedfrom ATCC). Approximately 5×10⁶ clones were plated from each of theaforementioned libraries, and screened under stringent hybridizationconditions employing radiolabeled full-length rat CART cDNA as ahybridization probe.

Hybridization positive cDNA clones were plaque purified, the cDNAinserts isolated in phagemid form following superinfection with helperphage, and the inserts subjected to complete nucleotide sequenceanalysis. The 5' end of human CART cDNA was determined by 5' RACE(GIBCO-BRL, Gaithersburg, Md.). Briefly, human hypothalamic poly(A+)mRNA was incubated with a complementary oligonucleotide to prime firststrand cDNA synthesis. The cDNA products were then tailed with terminaldeoxytransferase and dCTP, and subjected to PCR with appropriateoligonucleotides. The resulting cDNA fragments were subcloned andsubjected to full nucleotide sequence analysis.

For genomic DNA analysis, hybridization positive genomic DNA clones wereplaque purified, and phage DNA isolated. Eight unique oligonucleotidesspanning the entire human CART cDNA sequence were then utilized ashybridization probes for Southern blot analysis of the cloned genomicDNA fragments. A 2.5 kb SmaI/EcoRI genomic DNA fragment was shown tocontain the entire CART mRNA sequence following this analysis, and wassubsequently subjected to complete nucleotide sequence analysisfollowing the subcloning of appropriate, smaller DNA fragments into M13vectors, resulting in the complete sequence shown in SEQ ID No.:9. Allnucleotide sequence analysis was performed employing the Wisconsin CGCsuite of analytical software. The nucleotide sequence of full lengthhuman CART cDNA has been assigned accession number U16826 in the GenBankdatabase, with the human CART gene sequence assigned accession numberU20325.

PCR/Southern blot analysis of DNA isolated from 24 human/rodent somaticcell hybrids retaining one or two human chromosomes was used to localizethe human CART gene to a specific chromosome. DNA was analyzed fromNIGMS human/rodent somatic cell hybrid mapping panel #2. Included in thepanel was genomic DNA isolated from the human cell line IMR-91, thehamster cell line RJK88, and the mouse cell line 3T6. PCR was performedon all DNA samples using oligonucleotides directed against uniquesequences found in the 3' untranslated region of human CART mRNA (5'oligo to nucleotides 381-405, and 3' oligo to nucleotides 613-637,generating a PCR fragment 256 bp in length). The resulting PCR reactionswere then subjected to 1.5% agarose gel electrophoresis and Southernblot analysis employing full length, ³² P-radiolabeled human CART cRNAas a hybridization probe. The presence of a hybridization-positive, 256bp PCR-generated product indicates the presence of human genomic DNAencoding the CART transcript. Authenticity of this hybridization signalwas further shown by demonstrating that its appearance is dependent onthe presence of both 5' and 3' oligonucleotides in the original PCRreaction. The oligonucleotides employed for PCR reactions were directedagainst sequences found in the 3' untranslated region of human CART mRNA(5' oligo to nucleotides 381-405, and 3' oligo to nucleotides 613-637,generating a PCR fragment 256 bp in length). mRNA untranslated regionsare rarely conserved to a high degree between species, and for theaforementioned oligonucleotides this is clearly the case. Ahybridization-positive, PCR-generated DNA fragment of appropriate sizewas observed only with the human genomic DNA sample, with no such signalobserved for the corresponding murine and hamster DNA sample.Furthermore, generation of the human genomic DNA signal was dependent onthe presence of both 5' and 3' PCR oligonucleotides, supporting theauthenticity of the amplified DNA fragment. A similar hybridizationsignal was observed only in the human/rodent somatic cell hybrid samplecontaining human chromosome 5. No such signal was observed for the other23 DNA samples tested, and generation of the signal was also dependentupon the use of both 5' and 3' oligonucleotides in the PCR reaction. Anidentical analysis was performed on DNA from a different panel ofhuman/rodent somatic cell hybrids, and confirmed localization of thehuman CART gene to chromosome 5.

Example 5 Northern Blot Analysis of Human Brain Total RNA

Within the adult male rat, CART mRNA is present exclusively in neuraland endocrine tissues, as described above. Northern blot analysisrevealed that in the rodent brain, CART mRNA levels are highest withinthe hypothalamus, with midbrain/thalamus also exhibiting moderate levelsof expression. Lower CART mRNA levels are also observed in thehindbrain, hippocampus, striatum and cortex. The cerebellum appears tobe the only major brain structure devoid of detectable levels of CARTmRNA. In situ histochemical analysis further determined that within theaforementioned rodent brain regions, CART mRNA is found within specificneural circuits. For example, numerous limbic neural circuits expressthe CART transcript, including the amygdaloid complex, dentate gyrus ofthe hippocampus, hypothalamic mammillary and supramammillary nucleus,and nucleus accumbens. These sites of synthesis suggest a potentialfunctional role in reward processes and affect. Another predominantcircuit expressing major levels of the CART transcript is thatrepresented by hypothalamic neuroendocrine neurons, suggesting a rolefor the CART protein in regulation of endocrine events.

Observation of CART mRNA in similar human brain regions indicated thatCART plays a conserved functional role within the mammalian brain. Toprove this point, Northern blot analysis was performed on total RNAisolated from various regions of human brain, as follows. Human brain(with a negative drug toxicology profile) RNA (3-5 μg as determinedspectrophotometrically) was separated by electrophoresis on a 6%formaldehyde/1.2% agarose gel. The RNA was transferred to a nylonmembrane by capillary action with 20×SSC, followed by UV cross-linking.The membranes were then briefly dipped in 0.3 M sodium acetate, 0.02%methylene blue to stain the transferred RNA. 28S and 18S rRNA werereadily apparent, and served as a means by which to standardize for theamount of RNA contained within each sample. Following removal of stainby boiling in water for 10 minutes, the membranes were prehybridized for3-24 hr at 60° C. in hybridization buffer (5% SDS, 400 mM sodiumphosphate (7.0), 1 mM EDTA, 1 mg/mi BSA (fraction V) and 50% formamide).The prehybridization buffer was discarded and replaced with freshhybridization buffer including a full length, ³² P-radiolabeled humanCART cRNA probe. Hybridization proceeded for 16-24 hr at 42° C.Membranes were subsequently washed with 1% SDS, 0.05×SSC and 1 mM EDTAat 70°-75° C. for 1-4 hr, followed by exposure to Kodak XAR-5 film.

Rat eye total RNA served as a control sample to document the size of therat CART mRNA doublet (approximately 900 and 700 bases in length) whichresulted from the use of multiple poly(A) recognition sites (AATAAA).For the human brain RNAs examined, a major CART transcript was observedwhich was approximately 900 bases in length. This length was consistentwith the size and sequence of the human CART cDNA clones which wereanalyzed. Some samples revealed the presence of smaller species ofhybridization-positive mRNAs, which may be the result of alternatepoly(A) site utilization or RNA degradation. Major sites of synthesis ofthe human CART transcript were represented by the hypothalamus, frontalcortex and midbrain, with moderate to low levels observed in thehippocampus, motor cortex, and caudate-putamen. No observable CART mRNAwas detected in the cerebellum. Thus, the gross pattern of distributionof CART mRNA throughout the human brain is qualitatively similar to thatobserved for the rat, suggesting that the encoded CART protein plays aconserved functional role across mammalian species.

Example 6 CART Polysome Analysis

0.2 g of adult male Sprague-Dawley rat hypothalamus was homogenized in2.2 ml HKM buffer (100 mM NaCl, 20 mM Hepes (7.45), 1.5 mM MgCI₂, 0.5%Triton X-100, 2 mM vanadyl ribonucleotide complex) at 4° C. 0.2 ml wasremoved for experimental control purposes and stored at -20° C. Theremaining material was centrifuged at 9K rpm for 5 min at 4° C. in anSS34 rotor. The supernatant was removed and vortexed briefly. 1 ml wasremoved and loaded onto an approximately 11 ml, 10-35% sucrose gradientin 1×HMK buffer. Another 1 ml of supernatant was removed and adjusted to20 mM EDTA, and likewise loaded onto an approximately 11 ml, 10-35%sucrose gradient in 1×HMK buffer. The two sucrose gradients were thensubjected to ultracentrifugation at 37 krpm for 115 min at 4° C. in anSW41 rotor. Following centrifugation, the gradient was fractionated fromthe top into 1 ml aliquots (approximately 12 fractions were collected).RNA was then extracted from each gradient aliquot (as per proceduresroutine to our lab), and subjected to Northern blot analysis employing acRNA probe specific for the detection of rat CART mRNA.

Northern blot analysis was performed as described above. Rathypothalamic extracts either did not contain (-EDTA), or did contain(+EDTA) EDTA at a final concentration of 20 mM prior toultracentrifugation. In the gradient not containing EDTA, positivehybridization signals for rat CART mRNA were observed in fractions 8-12,with the majority of the hybridization signals observed in fractions9-11. This represented gradient fractionated material of a relativelyhigh density, as the material was found in the lower gradient fractions.RNA associated with polysomes is typically localized to this region of a10-35% sucrose gradient. Thus, this result suggests that in the rathypothalamus, CART mRNA is found on polysomes and is thus activelytranslated in vivo.

In the gradient containing EDTA, positive hybridization signals for ratCART mRNA were observed in fractions 2-4, with the majority of thehybridization signals observed in fractions 2 and 3. This representsgradient fractionated material of a relatively low density, as thematerial is found in the upper gradient fractions. Free RNA is typicallylocalized to this region of a 10-35% sucrose gradient. As the ability ofribosomes to interact with RNA is dependent on Mg₂, and EDTA serves tochelate (remove) Mg₂ from solutions, this result represents an expectednegative control.

Example 7 Production of Rat CART Fusion Proteins in Bacteria

pET15-rCART1

Rat CART1 protein was expressed as a fusion protein using the Novagenbacterial expression plasmid, pET15b. The coding region of rat CART1cDNA (excluding the predicted signal sequence) was amplified by PCRusing the following primers: ##STR1## The PCR product was isolated,purified, incubated with appropriate restriction enzymes and ligatedinto pET 15b, in accordance with the manufacturer's instructions.Ligations were transformed into E. coli, transformants isolated andresulting plasmid DNA analyzed by both restriction analysis andnucleotide sequence analysis. Nucleotide sequence analysis insured thatno errors in nucleotide sequence within the rCART1 coding region hadoccurred during the PCR reaction.

The resulting pET15b-rCART1 fusion protein had the following structure(underlined sequence represents that encoded by pET 15b, with thesequence shown in italics representing rCART1): ##STR2## where QED . . .(rCART1 ) . . . KCL represents the predicted mature rat CART proteinsequence, SEQ ID No.:6 (lacking the 13 amino acid alternativelytranslated insert SEQ ID No.:5).

The pET15b-rCART1 plasmid was then transformed in E. coli strain BL21(Novagen) for protein expression. 100 ml LB with 50 μg/ml Amp wasinoculated with 0.1 ml of an overnight culture and grown at 37° C. untilthe OD600 was 0.4-0.6. Samples were removed for SDS gel analysis andcultures were induced with 1 ml 100 mM IPTG. Cultures were incubated forthree additional hours at 37° C. Cultures were then harvested bycentrifugation, supernatants discarded and the cell pellet stored at-70° C. until fusion protein purification.

pET23b-rCART2

rCART2 was expressed as a fusion protein using the Novagen bacterialexpression system plasmid, pET23b. The coding region of rat CART2 cDNA(excluding the predicted signal sequence) was amplified by PCR using thefollowing primers: ##STR3## The PCR product was isolated, purified,incubated with appropriate restriction enzymes and ligated into pET23b.Ligations were transformed into E. coli, transformants isolated andresulting plasmid DNA analyzed by both restriction analysis andnucleotide sequence analysis. Nucleotide sequence analysis insured thatno errors in nucleotide sequence within the rCART2 coding region hadoccurred during the PCR reaction.

The resulting pET23b-rCART2 fusion protein had the following structure(underlined sequence represents that encoded by pET23b, with thesequence shown in italics representing rCART2): ##STR4## where QED . . .(rCART2) . . . KCL represents the entire rat CART sequence, SEQ IDNo.:4, minus the signal sequence.

The pET23b-rCART2 plasmid was then transformed in E. coli strain pLysS(Novagen) for protein expression. 100 ml LB with 50 μg/ml Amp and 34μg/ml chloramphenicol was inoculated with 0.1 ml of an overnight cultureand grown at 37° C. until the OD600 was 0.4-0.6. Samples were removedfor SDS gel analysis and cultures were induced with 1 ml 100 mM IPTG.Cultures were incubated for three additional hours at 37° C. Cultureswere then harvested by centrifugation, supernatants discarded and thecell pellet stored at -70° C. until fusion protein purification.

Purification of pET15b-rCART1 and pET23b-rCART2 fusion proteins

Frozen pET15b-rCART1 and pET23b-rCART2 bacterial pellets, prepared asdescribed above, were resuspended in 40 ml Binding Buffer (5 mMimidazole, 0.5 M NaCl, 20 mM Tris pH 7.5, 5 mM Beta- ME) and sonicatedin 5×30 sec bursts with chilling on ice between bursts. Lysates werecentrifuged at 10,000 rpm for 20 min at 4° C. and the supernatantsdiscarded. The pellets were resuspended in 20 ml Binding Buffer,resonicated and pelleted as described above. The pellets wereresuspended in 10 ml Binding Buffer +6 M Urea and chilled on ice for 60min. Lysates were centrifuged at 10,000 rpm for 20 min at 4° C., withthe supernatants removed and filtered through a 0.45 μm filter to removenon-resuspended particulate matter.

Affinity chromatography was then employed to purify the CART fusionproteins. The two fusion proteins contained the amino acid sequence-HHHHHH- (SEQ ID NO.:17) at either the amino- (pET15b-rCART1) orcarboxy- (pET23b-rCART2) terminus. No bacterial proteins are known tocontain this sequence. Properties unique to the HHHHHH (SEQ ID NO.:17)sequence allow for binding to the metal, nickel (Ni). Thus, a Ni- NTAagarose column (purchased from Qiagen) allowed for purification of theaforementioned CART fusion proteins to at least 95% purity as aredetermined by SDS-PAGE analysis.

2 ml of a 50% slurry of Ni-NTA agarose was mixed with 2 ml Milli-Q waterand allowed to settle in a column. The water was drained, and the resinrinsed with another 10 ml of Milli-Q water. The resin was thenequilibrated with 15 ml Binding Buffer +6 M urea. 10 ml of filtered celllysate from above was added to the column and mixed on a rotary shakerfor 60 min. The agarose resin was allowed to settle and the columndrained. The column was then washed sequentially with 3×5 ml BindingBuffer +6 M urea, and 3×7 ml Wash Buffer (20 mM imidazole, 0.5 ml NaCl,20 mM Tris pH 7.5, 5 mM Beta-ME, 6 M urea). The CART fusion proteinswere then eluted in 5 ml Elution Buffer (300 mM imidazole, 0.5 ml NaCl,20 mM Tris pH 7.5, 5 mM Beta-ME, 6 M urea).

The purification procedure was followed by SDS-PAGE analysis of aliquotsof crude bacterial lysates, column load material, column load flowthrough material, wash material and elution material. The final productwas judged to be at least 95% pure.

Stepwise dialysis was then performed to remove the urea from thepurified CART fusion proteins, and to allow the material to refold intoa proper native configuration. 2 ml of the purified protein wassequentially dialyzed for 45 min in 2×500 ml of 0.5 M NaCl, 20 mM TrispH 6.5, 0.1% Triton X-100 containing urea at concentrations of 4 M, 2 M,1 M, 0.5 M and 0 M. Additional dialysis was performed for 60 min in2×1000 ml 0.5 M NaCl, 20 mM Tris pH 6.5, 0.1% Triton X-100, 0.5%glycerol.

Example 8 Production of Human CART Proteins in Bacteria

The procedure of Example 7 is repeated substituting the coding region ofhuman CART cDNA (SEQ ID No.:7) for the rat CART cDNA. A human CARTfusion protein is obtained.

Example 9 Generation of Rabbit Polyclonal Antisera Against SyntheticCART Peptides

The following peptides, where underlined areas represent amino acidscontained within the rat CART amino acid sequence of SEQ ID No.:4, andnon-underlined residues (C and/or Y) represent residues placed at theN-terminus for purposes of enhanced antibody generation (C) orsubsequent radiolabeling with iodine (Y), were synthesized usingconventional techniques:

    ______________________________________                                        CQEDAELQPRALDIY                                                                              referred to as C-15-Y(SEQ ID No.: 18)                          CYRRQLRAPGAVLQ referred to as C-14-Q(SEQ ID No.: 19)                          CLKSKRIPIYEKKYG                                                                              referred to as C-15-G(SEQ ID No.: 20)                          YGARIGKLCDCPRGTSC                                                                            referred to as Y-l7-C(SEQ ID No.: 21)                          ______________________________________                                    

Polyclonal antisera to the synthetic peptides was produced usingconventional techniques, as follows. Primary (500 μg of syntheticpeptide) and secondary (250 μg of synthetic peptide) injections of eachsynthetic peptide were made into each of two female New Zealand whiterabbits on days 1 and 16, respectively, with boost injections (250 μg ofsynthetic peptide) being made on days 37, 58, 79 and 100, post primaryinjection. On days 47, 68, 89 and 110, post primary injection, eachanimal was bled and serum was obtained in a conventional manner.

The resulting antisera were designated as follows: Antibodies R1 and R2were raised against synthetic peptide C-14-Q; Antibodies R3 and R4 wereraised against synthetic peptide C-15-Y; Antibodies R5 and R6 wereraised against synthetic peptide C-15-G; and Antibodies R7 and R8 wereraised against synthetic peptide Y-17-C.

100 ng of affinity purified pET15b-rCART1 and pET23b-rCART2, prepared asdescribed in Example 7, were separated on a 15% SDS-polyacrylamide geland blotted onto nitrocellulose (Amersham) using semi-dryelectrophoretic transfer (Biorad). The blots were blocked overnight at4° C. in 5% non-fat dry milk in TBS-T (0.2% Tween/Tris-buffered saline).The blots were then rinsed with TBS-T followed by incubation for 2 hoursat room temperature in TBS-T including a 1:1000 dilution of one of theprimary antisera (R1-R8) or preimmune sera (serving as a negativecontrol). The blots were then rinsed with TBS-T and incubated for 1 hourin TBS-T containing a 1:2000 dilution of secondary antibody (goat anti-rabbit antibodies supplied as a horseradish peroxidase conjugate -Biorad). The blots were again rinsed with TBS-T, and any antigen-primary antibody-secondary antibody complex detected using the ECLdetection system (Amersham). Finally, the blots were exposed to KodakXAR-5 x-ray film for approximately 30 seconds to detect immunoreactiveproducts.

The presence of an approximately 14 kD immunoreactive band representingpET15b-rCART1, and an approximately 17 kD immunoreactive bandrepresenting pET23b-rCART2 indicated the presence of correspondingantibodies in the samples tested. For R6, no such signals were presentwhen preimmune sera was tested, while robust signals of appropriate sizewere observed when the immune sera was tested. Thus, R6 antiseracontained antibodies which detected both rCART1 and rCART2 fusionproteins, and represented an effective antisera. R3 and R7 antisera, onthe other hand, showed no difference between preimmune and immune sera,and were concluded to be of no value for specific recognition of rCARTproteins.

In addition, it was observed that in some Ab lanes, immunoreactivematerial was present representing higher molecular weight proteins. Itwas concluded that this material represents concatemerized material, inthat the observed molecular weight of this material is identical tomultiples (i.e., 2×, 3×, etc.) of the predicted rCART molecular weight.

Example 10 Production of Vaccinia Virus (VV) Expressing Rat CART

rCART1 (SEQ ID No.:6) and rCART2 (SEQ ID No.:4) cDNA, including theentire protein coding region as well as some additional 3' and 5'untranslated region, was subcloned into the plasmid pZVneo to facilitatetransfer and recombination into vaccinia virus, using the method of VanSlyke et al., supra, the disclosure of which is incorporated herein byreference. For this subcloning, PCR oligos were designed to engineer aBamHI restriction site onto the 5' end of the amplified cDNA, and anXhoI restriction site onto the 3' end of the amplified cDNA. Using fulllength rCART cDNAs in pBluescript SK- as templates, the cDNAs were PCRamplified employing Vent DNA polymerase (New England Biolabs), digestedwith BamHI and XhoI, and ligated into pZVneo. These resulting constructswere subsequently subjected to full nucleotide sequence analysisemploying a variety of oligonucleotide primers to ensure that no errorshad arisen within the resulting CART cDNAs during PCR amplification.These plasmid constructs were then transferred into vaccinia virus inBSC40 cells for production of high titer VV:rCART1 and VV:rCART2 virusvectors by the method of Van Slyke et al., supra.

Detection of rCART1 and rCART2 By Immunoprecipitation FollowingVV:rCART1 and VV:rCART2 Infection of Cultured Mammalian Cells

10 cm plates containing ˜10⁷ cells were infected at an MOI (multiplicityof infection) of 5 in PBS supplemented with BSA. BSC40 cells (Africangreen Monkey kidney cell line) were incubated with the VV recombinantsdescribed above for 30 min at room temperature. VV-containing media wasthen replaced with DMEM media supplemented with dialyzed 10% fetal calfserum (FCS), Gentamicin and 500μ Ci of EXPRESS protein labeling mix(i.e., 35S-Met and 35SCys, Dupont-NEN) for metabolic labeling of theVV-infected cells. The infected cells were then incubated overnight at37° C. Negative controls included mock infected cells (no VV) or cellsinfected with VV:furin to test for crossreactivity of CART antiseraagainst VV proteins. Culture media was removed and centrifuged for 10min at 3,000 rpm to clear cellular debris followed by storage at -20° C.The infected cells were rinsed in PBS, lysed on the plate in 1 ml coldlysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris pH 7.6), and thelysate collected into 1 ml Eppendorf tubes. The lysate was thencentrifuged for 20 min at 4° C., and the supernatants transferred tofresh tubes and stored at -20° C.

To detect radiolabelled rCART1 and rCART2 immunoprecipitation wasperformed. 300-500 μl of cell lysates and 900 μl of cell culture mediawere brought to a total volume of 1 ml with 1×lysis buffer or 10×lysisbuffer, respectively, followed by incubation with 100 μl ofrCART-specific antisera (or pre immune sera). The mixture was thenplaced on a rotating platform for 2 hours at 4° C. 30 μl of SepharoseA-agarose beads (Zymed) was then added, followed by incubation for anadditional 2 hours. The beads were then pelleted and washed 4 times with1 ml ice cold 1×lysis buffer. The beads were subsequently resuspended in30 μl of SDS sample buffer (60 mM Tris pH 6.8, 25% glycerol, 2% SDS,0.1% bromophenol blue, 0.7 M B-mercaptoethanol) and boiled for 5 min. 15μl of the sample was then subjected to electrophoresis on a 15% SDS-PAGEgel. Following electrophoresis the gels were fixed, followed byincubation in Amplify (Amersham) autoradiographic enhancement solution.The gels were then dried and subjected to autoradiographic analysis todetect the presence of radiolabeled, immunoprecipitated, 35Sradiolabeled rCART1 or rCART2.

For VV:rCART2 infected cells, an immunoprecipitated productapproximately 17 kD in size was observed in the cellular sample andmedia sample, indicative of rCART2. This product was not observed whenpreimmune sera was used. Likewise, for VV:rCART1 infected cells, animmunoprecipitated product approximately 14 kD in size was observed inthe cellular sample and media sample, indicative of rCART1 . The productwas not observed when preimmune sera was used. Thus, VV:CART infectedcells appear to make CART protein of predicted size, and furthermorethis material appears to be secreted into the media as predicted by thepresence of a signal sequence at the N-terminus of the predicted rCARTprotein. Additionally, these bands were not observed in both mockinfected cells (indicating that the aforementioned bands are not BSC40proteins), and VV:Furin infected cells (indicating that theaforementioned bands are not derived from the Vaccinia virus genome).

The foregoing procedure was repeated by incubating AtT20 cells with theVV recombinants for 2 hours at 30° C., in place of the BSC40 cells.Similar results were obtained. Thus, these results and conclusions arenot specific for BSC40 cells, but suggest that the deduced properties ofrCART are common to all mammalian cells expressing CART.

Pulse/Chase labeling of VV infected cells

As a further experiment, VV infections of AtT20 and BSC40 cells werecarried out as described above, with cells incubated overnight at 37° C.in complete DMEM supplemented with 10% fetal calf serum. Prior toradiolabeling, VV-infected cells were rinsed with and incubated incysteine- and methionine-deficient DMEM for 15 min. Cells were thenpulse-labeled for 45 min with cys/met free DMEM supplemented with 500μCi EXPRESS protein labeling mix. Labeling media was replaced withcomplete DMEM +10% FCS (the chase) and cells were harvested following a30 min, 90 min, 180 min, and 5 hour incubation period. Media and cellharvests were performed as described above. Radiolabeled proteins fromcell extracts (c) or media (m) were immunoprecipitated with R6 antisera.Immunoprecipitated products of approximately 17 kD represented CART2immunoreactive material, while immunoprecipitated products ofapproximately 14 kD represented CART1 immunoreactive material. For bothcell lines and VV recombinants tested, a time-dependent movement ofpulse-labeled CART from inside the infected cells to the culture mediawas observed. As time progressed, less CART material was present in thecells, and more material was found in the media. Thus, CART proteinswere determined to migrate from inside to cell to outside the cell in atime dependent manner, indicating that eukaryotic cells secrete CARTprotein, and that CART may exert its biological activity via release andsubsequent interaction with target cells. This finding confirms thatoriginally predicted by the presence of a hydrophobic signal sequence atthe amino-terminus of the predicted CART protein as deduced from theoriginal nucleotide sequence analysis. The additional 13 amino acids inrCART2 protein appear to have no effect on this biochemical property.

Patterns of Secretion of rCART1 and rCART2 Following Cell Stimulation

Two distinct types of secretory pathways are found in eukaryotic cells;regulated and constitutive release. Regulated release usually occurs incells containing dense-core secretory granules in which molecules arestored and released following some type of major stimulatory event.Constitutive release usually does not involve dense-core secretorygranules, and represents the means by which molecules are slowlyreleased from cells in a more uniform fashion. To determine how CARTmight be released from cells, use was made of the fact that AtT20 cellshave both constitutive and regulated secretory pathways, but BSC40 cellscontain only the constitutive secretory pathway. In the regulatorysecretory pathway, depolarization of the cell (such as by the additionof high extracellular potassium, K) causes stored material to bereleased in a huge bolus. Alternately, in the constitutive pathway,depolarization has no such effect. Thus, the effect of extracellularpotassium on AtT20 and BSC40 cells infected with VV:CART virus, the fateof CART proteins, and the type of secretory pathway through which CARTis released was determined as follows.

AtT20 or BSC40 cells were infected with VV:rCART1 or VV:rCART2, asdescribed above. Following infection, the cells were pulsed withradiolabeled amino acids for 45 min, followed by a 45 min chase. Thechase media (labeled as `chase`) was then collected for analysis, and asecond chase media added either containing (+) or not containing (-) KClfor an additional 10 min. After this incubation period, the cells andsecond chase media were collected for analysis.

Immunoprecipitated products of approximately 17 kD represented CART2immunoreactive material, while immunoprecipitated products ofapproximately 14 kD represented CART1 immunoreactive material. For bothcell lines and VV recombinants tested, KCl appeared to have no effect onthe levels of CART proteins present in either the cells or the secondchase media. If CART were present in secretory granules with releaseoccurring through the regulated secretory pathway, then KCl additionwould have been expected to increase levels of CART in the stimulatedmedia while decreasing levels of cellular CART. This was not observed.Rather, KCl addition had no apparent effect on media and cellular levelsof CART produced in both AtT20 cells and BSC40 cells. Thus, CART notonly appears to be secreted from mammalian cells, but secreted by meansof constitutive release. Also, the additional 13 amino acids in rCART2protein appear to have no effect on this biochemical property of theCART protein.

Example 11 Production of Vaccinia Virus Expressing Human CART

The procedure of Example 10 is repeated substituting human CART cDNA(SEQ ID No.:7) for the rat CART cDNA of Example 10. Human CART proteinis expressed by the infected cells, and similar results are obtained.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 21                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ACGTCTCATG10                                                                  (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GCTTTTTTTTTTT13                                                               (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 840 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: polyA.sub.-- site                                               (B) LOCATION: 548..553                                                        (D) OTHER INFORMATION: /note= "Poly(A) site for approximately                 700 base rat CART transcript"                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: polyA.sub.-- site                                               (B) LOCATION: 814..819                                                        (D) OTHER INFORMATION: /note= "Alternate Poly(A) site for                     approximately 900 base rat CART transcr..."                                   (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 20..409                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: primer.sub.-- bind                                              (B) LOCATION: 158..167                                                        (D) OTHER INFORMATION: /note= "5'PCR differential display                     primer hybridization site"                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: primer.sub.-- bind                                              (B) LOCATION: 544..556                                                        (D) OTHER INFORMATION: /note= "3'PCR differential display                     primer hybridization site"                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 179..217                                                        (D) OTHER INFORMATION: /note= "Alternatively spliced 39                       nucleotide sequence"                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AGCGAGGAAGTCCAGCACCATGGAGAGCTCCCGCCTGCGGCTGCTACCCGTC52                        MetGluSerSerArgLeuArgLeuLeuProVal                                             1510                                                                          CTGGGCGCCGCCCTACTGCTGCTGCTACCTTTGCTGGGTGCCGGTGCC100                           LeuGlyAlaAlaLeuLeuLeuLeuLeuProLeuLeuGlyAlaGlyAla                              152025                                                                        CAGGAGGATGCCGAGCTGCAGCCCCGAGCCCTGGACATCTACTCTGCC148                           GlnGluAspAlaGluLeuGlnProArgAlaLeuAspIleTyrSerAla                              303540                                                                        GTGGATGATGCGTCCCATGAGAAGGAGCTGCCAAGGCGGCAACTTCGG196                           ValAspAspAlaSerHisGluLysGluLeuProArgArgGlnLeuArg                              455055                                                                        GCTCCCGGCGCTGTGTTGCAGATTGAAGCGCTGCAGGAAGTCCTGAAG244                           AlaProGlyAlaValLeuGlnIleGluAlaLeuGlnGluValLeuLys                              60657075                                                                      AAGCTCAAGAGTAAACGCATTCCGATCTATGAGAAGAAGTACGGCCAA292                           LysLeuLysSerLysArgIleProIleTyrGluLysLysTyrGlyGln                              808590                                                                        GTCCCCATGTGTGACGCTGGAGAGCAGTGCGCAGTGCGGAAAGGGGCC340                           ValProMetCysAspAlaGlyGluGlnCysAlaValArgLysGlyAla                              95100105                                                                      AGGATCGGGAAGCTGTGTGACTGTCCCCGAGGAACTTCTTGCAATTCT388                           ArgIleGlyLysLeuCysAspCysProArgGlyThrSerCysAsnSer                              110115120                                                                     TTCCTCTTGAAGTGCTTGTGAAGGGGTGACAGCCTCCTTCGGTTCCCA436                           PheLeuLeuLysCysLeu                                                            125130                                                                        TATTTCCTCTTTCCCCTAAAGGAGCGCTCTTTTGTCCCTGGAGCCGCTTTAACAACAATA496               AAGTTTGCGTTCCCCCCAGAGAGTGGATGGGCTCTTTCCCTGCTGCTTCAAAATAAAAGA556               TTTGATGTTATTGTGTGAAGGACAATACCTTGAATGGTGTTGGTATGTGTGCAAAGTATT616               CTTCTCTCGTTTTATCCACCTGACACATTCTTGTGACCTTTCTGGGAAGAAGAGGGACTT676               TCGCTTTAAAACTGTATTTTTGTATGTGGCGGGTCACAATGAAGATTAGACCTAGTTAAT736               TTTGGCAGATGACATCATAACCCGGAAAACAAATCACCCCAAAGCAACACAAATGGAAGC796               ATGTGCAAATTACACCCAATAAAGCATTTTTGATAATTGCTCAC840                               (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 129 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetGluSerSerArgLeuArgLeuLeuProValLeuGlyAlaAlaLeu                              151015                                                                        LeuLeuLeuLeuProLeuLeuGlyAlaGlyAlaGlnGluAspAlaGlu                              202530                                                                        LeuGlnProArgAlaLeuAspIleTyrSerAlaValAspAspAlaSer                              354045                                                                        HisGluLysGluLeuProArgArgGlnLeuArgAlaProGlyAlaVal                              505560                                                                        LeuGlnIleGluAlaLeuGlnGluValLeuLysLysLeuLysSerLys                              65707580                                                                      ArgIleProIleTyrGluLysLysTyrGlyGlnValProMetCysAsp                              859095                                                                        AlaGlyGluGlnCysAlaValArgLysGlyAlaArgIleGlyLysLeu                              100105110                                                                     CysAspCysProArgGlyThrSerCysAsnSerPheLeuLeuLysCys                              115120125                                                                     Leu                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ProArgArgGlnLeuArgAlaProGlyAlaValLeuGln                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 116 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetGluSerSerArgLeuArgLeuLeuProValLeuGlyAlaAlaLeu                              151015                                                                        LeuLeuLeuLeuProLeuLeuGlyAlaGlyAlaGlnGluAspAlaGlu                              202530                                                                        LeuGlnProArgAlaLeuAspIleTyrSerAlaValAspAspAlaSer                              354045                                                                        HisGluLysGluLeuIleGluAlaLeuGlnGluValLeuLysLysLeu                              505560                                                                        LysSerLysArgIleProIleTyrGluLysLysTyrGlyGlnValPro                              65707580                                                                      MetCysAspAlaGlyGluGlnCysAlaValArgLysGlyAlaArgIle                              859095                                                                        GlyLysLeuCysAspCysProArgGlyThrSerCysAsnSerPheLeu                              100105110                                                                     LeuLysCysLeu                                                                  115                                                                           (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 800 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (A) DESCRIPTION: human CART cDNA (FIGURE 2)                                   (iii) HYPOTHETICAL: NO                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 20..370                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AACGACGAGTTTCAGAACGATGGAGAGCTCCCGCGTGAGGCTGCTGCCCCTC52                        MetGluSerSerArgValArgLeuLeuProLeu                                             1510                                                                          CTGGGCGCCGCCCTGCTGCTGATGCTACCTCTGTTGGGTACCCGTGCC100                           LeuGlyAlaAlaLeuLeuLeuMetLeuProLeuLeuGlyThrArgAla                              152025                                                                        CAGGAGGACGCCGAGCTCCAGCCCCGAGCCCTGGACATCTACTCTGCC148                           GlnGluAspAlaGluLeuGlnProArgAlaLeuAspIleTyrSerAla                              303540                                                                        GTGGATGATGCCTCCCACGAGAAGGAGCTGATCGAAGCGCTGCAAGAA196                           ValAspAspAlaSerHisGluLysGluLeuIleGluAlaLeuGlnGlu                              455055                                                                        GTCTTGAAGAAGCTCAAGAGTAAACGTGTTCCCATCTATGAGAAGAAG244                           ValLeuLysLysLeuLysSerLysArgValProIleTyrGluLysLys                              60657075                                                                      TATGGCCAAGTCCCCATGTGTGACGCCGGTGAGCAGTGTGCAGTGAGG292                           TyrGlyGlnValProMetCysAspAlaGlyGluGlnCysAlaValArg                              808590                                                                        AAAGGGGCAAGGATCGGGAAGCTGTGTGACTGTCCCCGAGGAACCTCC340                           LysGlyAlaArgIleGlyLysLeuCysAspCysProArgGlyThrSer                              95100105                                                                      TGCAATTCCTTCCTCCTGAAGTGCTTATGAAGGGGCGTCCATTCTCC387                            CysAsnSerPheLeuLeuLysCysLeu                                                   110115                                                                        TCCATACATCCCCATCCCTCTACTTTCCCCAGAGGACCACACCTTCCTCCCTGGAGTTTG447               GCTTAAGCAACAGATAAAGTTTTTATTTTCCTCTGAAGGGAAAGGGCTCTTTTCCTGCTG507               TTTCAAAAATAAAAGAACACATTAGATGTTACTGTGTGAAGAATAATGCCTTGTATGGTG567               TTGATACGTGTGTGAAGTATTCTTATTTTATTTGTCTGACAAACTCTTGTGTACCTTTGT627               GTAAAGAAGGGAAGCTTTGTTTGAAAATTGTATTTTTGTATGTGGCATGGCAGAATGAAA687               ATTAGATCTAGCTAATCTCGGTAGATGTCATTACAACCTGGAAAATAAATCACCCTAAGT747               GACACAAATTGAAGCATGTACAAATTATACATAATAAAGTGTTTTTAATAATT800                      (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 116 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetGluSerSerArgValArgLeuLeuProLeuLeuGlyAlaAlaLeu                              151015                                                                        LeuLeuMetLeuProLeuLeuGlyThrArgAlaGlnGluAspAlaGlu                              202530                                                                        LeuGlnProArgAlaLeuAspIleTyrSerAlaValAspAspAlaSer                              354045                                                                        HisGluLysGluLeuIleGluAlaLeuGlnGluValLeuLysLysLeu                              505560                                                                        LysSerLysArgValProIleTyrGluLysLysTyrGlyGlnValPro                              65707580                                                                      MetCysAspAlaGlyGluGlnCysAlaValArgLysGlyAlaArgIle                              859095                                                                        GlyLysLeuCysAspCysProArgGlyThrSerCysAsnSerPheLeu                              100105110                                                                     LeuLysCysLeu                                                                  115                                                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2483 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (A) DESCRIPTION: human CART genome DNA                                        (iii) HYPOTHETICAL: NO                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: promoter                                                        (B) LOCATION: 72..77                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: join(122..280, 706..789, 1329..1436)                            (ix) FEATURE:                                                                 (A) NAME/KEY: intron                                                          (B) LOCATION: 281..705                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: intron                                                          (B) LOCATION: 790..1328                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: polyA.sub.-- site                                               (B) LOCATION: 1581..1586                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: polyA.sub.-- site                                               (B) LOCATION: 1797..1802                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: polyA.sub.-- site                                               (B) LOCATION: 1846..1851                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CCCGGGCCCTCCTCCACCCCCCCTTCCTTCTTCGCCTCCTCCCTCTTTCCTGCACGGGGG60                CTCGGGCTCACTATAAAAGGTGGGAGCGCGTGGTGCCCCAGCAACGACGAGTTTCAGAAC120               GATGGAGAGCTCCCGCGTGAGGCTGCTGCCCCTCCTGGGCGCCGCC166                             MetGluSerSerArgValArgLeuLeuProLeuLeuGlyAlaAla                                 151015                                                                        CTGCTGCTGATGCTACCTCTGTTGGGTACCCGTGCCCAGGAGGACGCC214                           LeuLeuLeuMetLeuProLeuLeuGlyThrArgAlaGlnGluAspAla                              202530                                                                        GAGCTCCAGCCCCGAGCCCTGGACATCTACTCTGCCGTGGATGATGCC262                           GluLeuGlnProArgAlaLeuAspIleTyrSerAlaValAspAspAla                              354045                                                                        TCCCACGAGAAGGAGCTGGTCGGTATTCCCCTCGCTCTCGACCCCCTT310                           SerHisGluLysGluLeu                                                            50                                                                            GACGTGTCGCCTTGTCTCTTCTCTTGCACGCCTCCCTCCTTCCCCCCACCCCCACTCCTA370               TTCCCAGAGTCAGGGCGCGGGGAGCTGAGCGCAACGCCCAGGCACCCACTGCCATCCGAA430               GAGCGACTCGAGCTCACGGGCTCCTGGCAGTCTGTTGAGCGAATCCCTCATCCCGGCCCC490               TCTGAGCAACAGGGGCCCCAGCGGCTCAGAGACCCGCGGTCAGTACCTGGGACAGCGTCG550               CTAAGTTTCCACCCCTCGACCATTCCCTGTGTCCGCGGAGTCCCACCGCAGAGTGCGTGT610               GGGTCCGGGGCTCCTTATAACTAGGGCTGGAAGTGCGCACCTGGGCTGGGCTCGCAGCAA670               GGCGCAACTTCAGGCTCCGAAGCGGTGTGTTGCAGATCGAAGCGCTGCAAGAA723                      IleGluAlaLeuGlnGlu                                                            55                                                                            GTCTTGAAGAAGCTCAAGAGTAAACGTGTTCCCATCTATGAGAAGAAG771                           ValLeuLysLysLeuLysSerLysArgValProIleTyrGluLysLys                              60657075                                                                      TATGGCCAAGTCCCCATGGTAAGGTTTGTGGTCACTCCCTTCCCGTGT819                           TyrGlyGlnValProMet                                                            80                                                                            TTTTCCAAGAGAAAGTACACCGCCTTGAATCGTACACACAGCTCCGTAGGATGTGGCTAA879               ATAACTTAGGTAATGGGCTTGCAGGATTCTGTGGGCTCCTTCTTCCTTCCCGGGTGAGGA939               AATGGGAAAGCAGGAACAGGGGTTGTAAGAAAGTGTAAGTCTATTGTTTGTTGCTCAGGA999               AAAAGGTCTGATTTTTTTCCCTCTGAGAGGGCAAGAAAAGGAGCCAGGAAATGTGATGCT1059              CCCCTTCCCACGCCCCCCAACCCTCGCCACTTAAAGGTGGAAGAAACTAGGATAAAACTA1119              ATAATGTAAGTTTCTTTAAAAAATGTACTCTCACTGAGGTTATAAGCACAAGGCTCCCTG1179              TTTCAGATCTGACTGTACGTCGACCTCTTGTGATGGTGATGGGGTCCAATTGCCCCTTTC1239              AAGAGACAGAAATTGCGTTGACTGTGAGACTTGCCTGTTGGGAACCTGGGTTTGTTCATA1299              CTCGATGACCACACATTTTGTTGTTTCAGTGTGACGCCGGTGAGCAGTGTGCA1352                     CysAspAlaGlyGluGlnCysAla                                                      85                                                                            GTGAGGAAAGGGGCAAGGATCGGGAAGCTGTGTGACTGTCCCCGAGGA1400                          ValArgLysGlyAlaArgIleGlyLysLeuCysAspCysProArgGly                              9095100105                                                                    ACCTCCTGCAATTCCTTCCTCCTGAAGTGCTTATGAAGGGGCGTCCATTCTCC1453                     ThrSerCysAsnSerPheLeuLeuLysCysLeu                                             110115                                                                        TCCATACATCCCCATCCCTCTACTTTCCCCAGAGGACCACACCTTCCTCCCTGGAGTTTG1513              GCTTAAGCAACAGATAAAGTTTTTATTTTCCTCTGAAGGGAAAGGGCTCTTTTCCTGCTG1573              TTTCAAAAATAAAAGAACACATTAGATGTTACTGTGTGAAGAATAATGCCTTGTATGGTG1633              TTGATACGTGTGTGAAGTATTCTTATTTTATTTGTCTGACAAACTCTTGTGTACCTTTGT1693              GTAAAGAAGGGAAGCTTTGTTTGAAAATTGTATTTTTGTATGTGGCATGGCAGAATGAAA1753              ATTAGATCTAGCTAATCTCGGTAGATGTCATTACAACCTGGAAAATAAATCACCCTAAGT1813              GACACAAATTGAAGCATGTACAAATTATACATAATAAAGTGTTTTTAATAATTGCCCATA1873              GTGCACTGCTGTTTTCATATAAGTAATTTAAGTGGAAATGGTGAGATTAATCATGCTGTT1933              GTTTTCAAAGAAAAATATTTCAAAAATAGCAGCCTATTGGAAATGCACTACGTCAGAGTT1993              GATCGTATAGAGTTGCAGCAGTTAGTATACCTATTTCTTGATGCAGCGAGTGTGTGTGTA2053              TGTGTGTGTGTTAGTGTGTGTGTGTGTGTGTGTGTGAGAGAGAGAGAGAGAGAGAAAGAG2113              AGAGATGAATGAGATGGAGATGGTTGGAGAAGAGGTTATATAATTTTGTTTATTAAAACC2173              TTTAGCCAGACCCTTTACTTTAAACAGTGAGACCAATAAACTATAAACAGTTTCATGTTT2233              TAGTCACATTAAAAGCAATTTGAAAAATTAGAAATTTTGTTTTGACAACTCCCTTATTAG2293              AAAATATACATTGATTTAAAGATATGGGCTGTTTAGGGTTGTTATTTGTCTAAAGACTCC2353              AAGGTTATAAGACCCATCCATCCCACAAGTAAATTCACACTCTTGGAAAAATTCTCTATT2413              CCAGGAGAAAGAGTCATTTCAGAAAATAGTTTTGAGGGGAACAAATAAAAATTGGAGGAG2473              GTGAGAATTC2483                                                                (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (A) DESCRIPTION: N-terminal rCART1 cDNA primer                                (Example7)                                                                    (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CGATCGGCATATGCAGGAGGATGCCGAGCTG31                                             (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (A) DESCRIPTION: C-terminal rCART1 cDNA primer                                (Example7)                                                                    (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CGGCGGATCCGTTAAAGCGGCTCCAGGG28                                                (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (A) DESCRIPTION: N-terminal rCART2 cDNA primer                                (Example7)                                                                    (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GGTCGGGATCCGCAGGAGGATGCCGAGCTG30                                              (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (A) DESCRIPTION: C-terminal rCART2 cDNA primer                                (Example7)                                                                    (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GGTGCTCGAGCAAGCACTTCAAGAGGAAAG30                                              (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (A) DESCRIPTION: Kozak translation initiation sequence element                (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:\:                                    GCCGCCRCCATGG13                                                               (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 110 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (A) DESCRIPTION: pET15b-rCART1 fusion protein                                 (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      MetGlySerSerHisHisHisHisHisHisSerSerGlyLeuValPro                              151015                                                                        ArgGlySerHisMetGlnGluAspAlaGluLeuGlnProArgAlaLeu                              202530                                                                        AspIleTyrSerAlaValAspAspAlaSerHisGluLysGluLeuIle                              354045                                                                        GluAlaLeuGlnGluValLeuLysLysLeuLysSerLysArgIlePro                              505560                                                                        IleTyrGluLysLysTyrGlyGlnValProMetCysAspAlaGlyGlu                              65707580                                                                      GlnCysAlaValArgLysGlyAlaArgIleGlyLysLeuCysAspCys                              859095                                                                        ProArgGlyThrSerCysAsnSerPheLeuLeuLysCysLeu                                    100105110                                                                     (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 124 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (A) DESCRIPTION: pET23b-rCART2 fusion protein                                 (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      MetAlaSerMetThrGlyGlyGlnGlnMetGlyArgAspProGlnGlu                              51015                                                                         AspAlaGluLeuGlnProArgAlaLeuAspIleTyrSerAlaValAsp                              202530                                                                        AspAlaSerHisGluLysGluLeuProArgArgGlnLeuArgAlaPro                              354045                                                                        GlyAlaValLeuGlnIleGluAlaLeuGlnGluValLeuLysLysLeu                              505560                                                                        LysSerLysArgIleProIleTyrGluLysLysTyrGlyGlnValPro                              65707580                                                                      MetCysAspAlaGlyGluGlnCysAlaValArgLysGlyAlaArgIle                              859095                                                                        GlyLysLeuCysAspCysProArgGlyThrSerCysAsnSerPheLeu                              100105110                                                                     LeuLysCysLeuLeuGlyHisHisHisHisHisHis                                          115120                                                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      HisHisHisHisHisHis                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CysGlnGluAspAlaGluLeuGlnProArgAlaLeuAspIleTyr                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CysTyrArgArgGlnLeuArgAlaProGlyAlaValLeuGln                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      CysLeuLysSerLysArgIleProIleTyrGluLysLysTyrGly                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      TyrGlyAlaArgIleGlyLysLeuCysAspCysProArgGlyThrSer                              151015                                                                        Cys                                                                           -47-                                                                          __________________________________________________________________________

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A purified and isolatedCART nucleic acid comprising a nucleotide sequence selected from thegroup consisting of:a) a nucleotide sequence as shown in FIG. 1 (SEQ IDNo.:3), FIG. 2 (SEQ ID No.:7), or FIG. 3 (SEQ ID No.:9); b) a nucleotidesequence which hybridizes under stringent conditions to a nucleotidesequence defined in (a) wherein stringent hybridization conditionscomprise incubation at 42° C. in hybridization buffer comprising 400 mMsodium phosphate (pH 7.0), and 50% formamide; and c) a nucleotidesequence encoding the same polypeptide as encoded by the nucleotidesequences of (a) or (b) by means of degenerate codons.
 2. A nucleotidesequence of claim 1 which comprises a cDNA sequence that encodes thehuman CART polypeptide of SEQ ID No.:8 or the rat CART polypeptide ofSEQ ID No.:4 or SEQ ID No.:6.
 3. A nucleotide sequence of claim 2 whichis selected from the group consisting of the sequence of SEQ ID No.:7and SEQ ID No.:3.
 4. A nucleotide sequence of claim 1 which comprises agenomic sequence that encodes the human CART polypeptide of SEQ ID No.:8or the rat CART polypeptide of SEQ ID No.:4.
 5. A nucleotide sequence ofclaim 4 which comprises the sequence of SEQ ID No.:9.
 6. A procaryoticor eucaryotic host cell transformed or transfected with a nucleotidesequence according to claim
 1. 7. A viral or circular plasmid comprisinga DNA sequence according to claim
 1. 8. A viral or circular DNA plasmidaccording to claim 7 further comprising an expression control DNAsequence operatively associated with said CART encoding DNA.
 9. A hostcell comprising a plasmid of claim
 7. 10. A host cell comprising aplasmid of claim 8.